Citation
Protein targeting and translocation on cpSec and Delta pH pathways in chloroplast thylakoids

Material Information

Title:
Protein targeting and translocation on cpSec and Delta pH pathways in chloroplast thylakoids
Creator:
Ma, Xianyue, 1958-
Publication Date:
Language:
English
Physical Description:
xi, 95 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Chlorophylls ( jstor )
Chloroplasts ( jstor )
Clines ( jstor )
Imports ( jstor )
In vitro fertilization ( jstor )
Lumens ( jstor )
Protein transport ( jstor )
Receptors ( jstor )
Thylakoids ( jstor )
Dissertations, Academic -- Plant Molecular and Cellular Biology -- UF ( lcsh )
Plant Molecular and Cellular Biology thesis, Ph.D ( lcsh )
Plant proteins -- Physiological transport ( lcsh )
Protein binding ( lcsh )
Thylakoids ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1999.
Bibliography:
Includes bibliographical references (leaves 85-94).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Xianyue Ma.

Record Information

Source Institution:
University of Florida
Rights Management:
The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
030361454 ( ALEPH )
41876956 ( OCLC )

Downloads

This item has the following downloads:


Full Text









PROTEIN TARGETING AND TRANSLOCATION ON cpSEC AND DELTA pH
PATHWAYS IN CHLOROPLAST THYLAKOIDS











By

XIANYUE MA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1999














ACKNOWLEDGMENTS


The author thanks his supervisor, Dr. Kenneth Cline, for providing an exceptional environment where the author received full training in both research skills and scientific thinking. The author appreciates the advice and care from the members of his committee Drs. Kenneth Cline, Alice Harmon, Alfred S. Lewin, Daryl R. Pring, and Carlos Eduardo Vallejos. The author especially thanks Dr. Ralph Henry for his kind help in the author's experimental operations. Dr. Henry's early work with DT23 also helped the author's research project. The author wants to thank Dr. Baocai Tan and Wentao Deng for their help in DNA work such as tOE17 subcloning and expression. Thanks go to Dr. Hiroki Mori for providing antibodies. The author also wishes to thank Mike McCaffery, Vivian Fincher, Shaw Wu, and Dr. Liz Summer for their help. This dissertation is dedicated to the author's 81-year old mother Yisong Wu for asking her forgiveness for her son's years of absence.















TABLE OF CONTENTS


paIge

ACKNOW LEDGM ENTS..................................................................................................... ii

KEY TO ABBREVIATION S.......................................................................................... iv

ABSTRACT ......................................................................................................................... v

CHAPTERS

1 LITERATURE REVIEW .................................................................................................. 1

Protein Import into Chloroplast Stroma ............................................................................. 2
Protein Transport into Thylakoids ..................................................................................... 5
Summary and Perspective................................................................................................ 18

2 PROTEIN TARGETING AND TRANSLOCATION ON THE cpSec PATHWAY.........22

Abstract ...........................................................................................................................22
Introduction.....................................................................................................................23
M aterials and M ethods...................................................................................................25
Results. ...........................................................................................................................29
Discussion.......................................................................................................................46

3 TARGETING AND TRANSLOCATION ON THE DELTA pH PATHWAY................54

Abstract ........................................................................................................................... 54
Introduction ..................................................................................................................... 55
M aterials and M ethods...................................................................................................56
Results. ........................................................................................................................... 59
Discussion .......................................................................................................................79

4 SUM M ARY AND CON CLUSIONS............................................................................... 83

REFERENCES ................................................................................................................... 85

BIOGRAPHICAL SKETCH .............................................................................................. 95














KEY TO ABBREVIATIONS


APDP N-[4-(p-Azidosalicylamido) butyl]-3'-[2'-pyridyldithio] propionamide BS3 bis [sulfosuccinimidyl] suberate CCCP carbonylcyanide 3-chlorophenylhydrazone DSS disuccinimidyl suberate DTT dithiothreitol LTD lumen targeting domain LHCP light-harvesting chlorophyll a/b protein MBS m-Maleimidobenzoyl-N-hydroxysuccinimide ester OE17 the 17-kDa subunit of the oxygen-evolving complex OE23 the 23-kDa subunit of the oxygen-evolving complex OE33 the 33-kDa subunit of the oxygen-evolving complex Pmf proton motive force PC plastocyanin SE stromal extract STD stroma targeting domain














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

PROTEIN TARGETING AND TRANSLOCATION ON cpSEC AND DELTA pH
PATHWAYS IN CHLOROPLAST THYLAKOIDS By

XIANYUE MA

May 1999

Chair: Kenneth C. Cline
Major Program: Plant Molecular and Cellular Biology


Protein transport is required for biogenesis of the chloroplast, the site of photosynthesis. There are at least two pathways for protein transport into the chloroplast thylakoid lumen. One pathway is named the cpSec pathway, another is named the Delta pH pathway. Each pathway is specific for a subset of precursor proteins. I hypothesize that targeting, a specific interaction between the precursor and components of the protein transport machinery, commits precursor proteins to a specific pathway.

I have attempted to describe and characterize the first committed step on the cpSec pathway and to define in general terms the sequences required for commitment. Binding of precursors to the thylakoid membrane appears to be the first committed step. It required stromal protein, of which cpSecA was the essential component, and occurred when transport was inhibited by removing ATP from the assay with apyrase. Subsequent transport of bound precursor required ATP, but was not stimulated by additional cpSecA.








My results show that precursor binding to the membrane results in formation of a large complex that includes precursor, cpSecA, and cpSecY. The complex formation with cpSecA is precursor specific, requiring a cpSec transit peptide and additionally a cpSeccompatible flanking region.

Techniques were developed to dissect protein transport on the Delta pH pathway into binding (targeting) and chase (translocation). A newly constructed precursor protein tOE17 that uses the Delta pH pathway, bound to the membrane in the absence of the ApH. Binding is productive because bound precursor was transported into the lumen when the ApH was restored. Productive binding was competed by saturating amounts of a Delta pH pathway precursor protein, suggesting that precursor tOE17 binds to components of the Delta pH pathway translocation machinery. Consistent with the transport requirement, the ApH was the only energy source used for chase. Interestingly, the chase of bound precursor was also competed by over expressed precursor. Antibodies raised to E. coli-expressed Hcfl106 specifically inhibited tOE17 transport and reduced the level of productive binding. My results suggest that Hcfl06 functions in early steps of the transport process, possibly as a receptor.














CHAPTER 1
LITERATURE REVIEW


Although the sun is the primary energy source for all life, it is in chloroplast that the light energy from the sun is converted into biologically usable chemical energy. Apart from photosynthesis, several other important biosynthetic processes also occur in chloroplasts, such as biosynthesis of amino acids, lipids, pigments and plant hormones.

A mature chloroplast can be spatially divided into six compartments: outer envelope, inner envelope, interenvelope space, stroma, thylakoid membrane and thylakoid lumen. The chloroplast is enclosed by the envelopes; inside the chloroplast the thylakoid stacks float in the stroma. The well differentiated chloroplast develops from a small, more or less undifferentiated proplastid. This morphogenetic process is controlled by endogenous and exogenous factors and involves a massive production of proteins that are required for the functions of the chloroplast. The chloroplast is a semi-autonomous organelle. It contains genetic material and has functional systems for both gene transcription and translation. However, its circular DNA molecule contains only about 100 genes, about 69-77 of which encode proteins (Sugiura et al. 1998). More than three fourth of proteins residing in the chloroplasts are nucleus encoded and synthesized in the cytoplasm. These proteins must be imported into the chloroplast from the cytosol. It is obvious that protein import plays an important role in chloroplast biogenesis.








Protein Import into Chloroplast Stroma

Significant advances in understanding chloroplast protein transport began two decades ago when protein import was reconstituted with isolated chloroplasts (Chua and Schmidt 1978, 1979). The in vitro or in organello transport assay is conducted by mixing radiolabeled precursor protein with purified chloroplasts. Active chloroplasts for protein import are prepared by homogenization of fresh young green tissues followed by Percoll gradient centrifugation and isotonic buffer wash. Radiolabeled precursor proteins are prepared by in vitro translation of mRNA with a wheat germ system (Cline et al. 1989) or rabbit reticulocyte system in the presence of one radiolabeled amino acid. Large amounts of precursor protein can be made by expression of the protein in E. coli and subsequent purification (Weisbeek et al. 1989; Pilon et al. 1990; Waegemann 1990)



Stroma Targeting Domain

A nuclear-encoded chloroplast protein is initially synthesized in the cytosol as a precursor protein that contains a transient transit peptide at its amino terminus (Dobberstein et al. 1977). For stroma-targeted precursor the transit peptide consists of a stromal targeting domain (STD) that governs the import of the precursor across the chloroplast envelope. Once in stroma the precursor stromal targeting domain is cleaved by a processing protease (Robinson and Ellis 1984, Abad et al. 1989 VanderVere et al. 1995).

Stromal targeting domains range from 30 to 120 amino acid residues and are predicted to have a random coil conformation (von Heijne and Nishikawa 1991). Although the stromal targeting domains from different precursors are interchangeable for








targeting passenger proteins across the chloroplast envelope, there are no conserved sequence blocks among the stromal targeting domains. However, stromal targeting domains are characterized to share three compositional motifs: a 10-15 residue Nproximal portion without charged residues, glycine or proline; a variable middle region that lacks negative charged residues and is rich in hydroxylated amino acids (serine and threonine); and a C-terminal region that contains a loosely conserved sequence (Ile/ValX-Ala/Cys*Ala) for proteolytic processing (von Heijne et al. 1989, de Boer and Weisbeek 1991)



The Import Process

Protein import across chloroplast envelope into the stroma is considered to be a three step process (Cline and Henry 1996, Schnell 1998, Soil and Tien 1998). The first is reversible precursor binding to the outer envelope. This step does not require any energy. The binding is achieved via the interaction of the precursor stromal targeting domain with proteinaceous receptors and possibly polar lipids at the surface of outer membrane (Cline et al. 1985). The translocon at the outer chloroplastic envelope membrane (Toe complex) contains three integral membrane proteins: Toc34, Toc86 and Toc75. Two lines of evidences suggest that Toc86 and Toc34 function as primary receptors: first, crosslinking studies indicate that Toc86 and Toc34 are contact with the precursor during the initial binding (Perry and Keegstra 1994, Ma et al. 1996, Kouranov and Schnell 1997); second, anti-Toc86 IgGs block the binding of precursor to the envelope (Hirsch et al. 1994).








In the second step of import, the precursor inserts across the outer envelope, resulting in close physical proximity to Toc75 (Perry and Keegstra 1994, Ma et al. 1996). Toc75 is deeply embedded in the outer envelope (Schnell et al. 1994, Tranel et al. 1995). Analysis of the Toc75 sequence indicates that it may traverse the membrane with 16 hydrophobic P-strands. Toc75 is therefore proposed to function as a protein translocation channel and this was verified in vitro by studies using expressed Toc75 reconstituted into liposomes (Hinnah et al. 1997). Unlike the first step of import in which the binding is energy independent and reversible, the second step requires the hydrolysis of both ATP and GTP (less than 100 ptM) and results a tight association of the precursor with the Toc complex (Kessler et al. 1994, Olsen and Keegstra 1992, Ma et al. 1996). Precursor insertion across the outer envelope also brings the transit peptide (stromal targeting domain) near to the translocon at the inner chloroplastic envelope membrane (Tic complex) (Schnell and Blobel 1993). In their in vitro binding assay, Friedman and Keegstra (1989) and Schnell and Blobel (1993) estimated that there are about 1500-3000 import sites per chloroplast. However, immunogold labeling of Toc34 in cryosubstituted thin sectioned chloroplasts suggested that there were at least 15000-20000 import sites (Morin and Soil 1997). The difference indicates that in the isolated chloroplasts only a small percentage of import sites are functional for precursor binding or import, implying that only receptors in a certain state can be used. Both Toc86 and Toc34 are GTP-binding proteins (Kessler et al. 1994). It is possible that GTP status may determine receptor state.

Several proteins have been identified as components of the translocon at the inner chloroplastic envelope. They are Ticl4, Tic21, Tic22, Tic55, TicI 10 etc. Ticl4, Tic21 and Tic22 were identified by cross-linking to a precursor protein (Ma et al. 1996,








Kouranov and Schnell 1997). Tic22 is peripherally localized at the out face of the inner envelope and considered to be an inner envelope receptor. Tic 1l0 was found associated with two stroma chaperones, cpn60 and ClpC; it may be involved in driving precursor transport or in folding of newly imported proteins in the stroma (Kessler and Blobel 1996). Tic55 was found associated with Tic 10 and the Toc components Toc86, Toc34 and Toc75 in both blue-native gel electrophoresis and affinity chromatography assays (Caliebe et al. 1997). Tic55 contains an iron sulfur cofactor. Soil and Tien (1998) hypothesized that Tic55 may act as a regulatory factor by using the iron-sulfur cluster as a redox sensor to influence protein transport.

The third step of import is protein translocation into the stroma. This step requires the cooperation of both Toc and Tic complexes. The precursor is transported across both outer and inner envelopes via the contact sites (Schnell and Blobel, 1993). ATP hydrolysis at higher concentration (>100 pM) in the stroma is used as energy source (Pain and Blobel 1987, Theg et al. 1989). The stromal targeting domain is cleaved by a soluble processing peptidase during or immediately following protein import into the stroma, creating a mature stromal protein (Robinson and Ellis 1984, Abad et al. 1989, VanderVere et al. 1995).



Protein Transport into Thylakoids

Proteins destined for the thylakoid lumen have a bipartite transit peptide: the aminoproximal region is a stroma-targeting domain (STD); the STD is followed by a lumentargeting domain (LTD) (Ko and Cashmore 1989, Hageman et al. 1990). The bipartite nature of the transit peptide was first proposed by Smeekens et al. (1986) based on their








assay with chimeric precursor proteins. The precursor PC transit peptide not only directed the precursor into the chloroplast stroma but also targeted the mature PC into its subsequent localization. Import of the precursor across the chloroplast envelope is governed by the STD of the transit peptide and the import process is the same as described above for a nucleus-encoded stromal protein. The STD is cleaved by a processing protease in the stroma, exposing the LTD and giving rise to an intermediate precursor protein. The LTD further directs transport into the thylakoid lumen where the LTD is removed by a second processing protease, thereby producing the mature protein (Halpin et al. 1989).

In vivo and in organello, the precursor imports across the chloroplast envelope first and then transports into the thylakoid lumen. However, import and transport are independent processes. With in vitro assays, precursor can be directly transported into the thylakoids bypassing the import step (Cline 1986, Kirwin et al. 1989). A stromal intermediate precursor was accumulated when thylakoid transport was inhibited during a chloroplast import assay (Cline et al. 1989). The intermediate was in a productive state and it could be chased into the thylakoids once the transport inhibition was removed (Cline et al. 1993). The plastid-encoded thylakoid protein cytochrome f is synthesized with only a LTD, its transport should not have any relation with the import process (Nohara et al. 1996, Zak et al. 1997).

Actually, full-length precursor with both STD and LTD are efficient substrates for transport into isolated thylakoids (Robinson and Klosgen 1994). This implies that thylakoid protein transport employs a loop mechanism similar to that used by the E. coli Sec system and the endoplasmic reticulum transport system (Shaw et al. 1988, Kuhn et








al. 1994). This hypothesis was clearly proven by Fincher et al. (1998). The topology of precursor insertion into the thylakoid membrane was investigated with a fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17. While the mature OE17 was transported to the thylakoid lumen, the amino terminus, including the pOE17 transit peptide, remained on the cis side of the thylakoid membrane.

Although there appears to be one major pathway for protein import across the chloroplast envelope, there are at least four pathways for protein transport into the thylakoid lumen or integration in the thylakoid membrane (Figure 1-1). Multiple pathways for thylakoid protein transport were first recognized by the discovery that different subgroups of precursors had different energy and soluble protein factor requirements for translocation (Cline et al. 1992). The existence of pathway specific translocation machinery was further demonstrated by precursor competition studies (Cline et al. 1993). For example, saturating concentration of iOE23 inhibited transport of OE17, OE23 and PSIIT, but was without effect on transport of OE33 and PC.

One of the thylakoid transport pathways is called the cpSec pathway since this pathway employs a chloroplast homologue of the bacterial SecA protein. A second pathway is named the Delta pH pathway because in this pathway, the ApH across the thylakoid membrane is the only energy requirement for protein transport. The third is the cpSRP pathway and the fourth is the spontaneous pathway. Each pathway is specific for a subset of precursor proteins (Cline and Henry 1996, Schnell 1998, Robinson et al. 1998).

In this dissertation, research focuses on protein transport on the cpSec and Delta pH pathways. Literature about protein transport on these two pathways will be reviewed in the following sections.























pre-lumenal pLHCP protein


pre-membrane
protein


outer envelope
z


In


cp8RP

SIPFc OE23
IO 3 JOE17 . .
"'.. 1sF IpSrr
IP81N Ps2W
GVTP+ Ps2X ATP+ mA pH tion. pH PH oft only


nor envelope lurmen


thylakold membrane


Figure 1-1. Schematic representation of the one general pathway for protein import across chloroplast envelope and four known pathways for protein transport to the thylakoids (From Keegstra and Cline, 1999).








The LTD Structure and Precursor Targeting

The LTD can be divided into four sub-domains based on the nature of the amino acid residues (Cline and Henry 1996): an acidic (A) domain of about 12-15 amino acid residues is on the amino-terminus of the LTD; this is followed by a positively charged

(N) domain of about 4-7 amino acid residues; then a hydrophobic (H) domain of 12-18 amino acid; and a C-terminal (C) domain with a relatively conserved motif of Ala-X-Ala for proteolytic processing. Figure 1-2 shows some examples of LTDs that confer either cpSec or Delta pH pathway specifically. Also shown is an artificially produced LTD that confers transport on both pathways.

The N, H and C domains of LTD represent the basic structure of the classical signal peptides that direct proteins to the endoplasmic reticulum or the bacterial plasma membrane. This implies that the LTD is evolutionarily related to these signal peptides and that thylakoid protein transport is conserved from the original bacterial endosymbiont (von Heijne et al. 1989). Additional support for the conservative origin of LTDs came from the fact that LTD is cleaved by a thylakoid lumenal processing peptidase (TPP) with identical specificity to a bacterial leader peptidase (LPP), which can carry out faithful processing if the protein is expressed in E. coli and exported across bacterial inner membrane (Halpin et al. 1989, Anderson et al. 1991). Thylakoid proteins OE33 and plastocyanin, as well as DT23 with a dual targeting transit peptides, were exported to the periplasmic space and properly processed to their mature sizes when expressed in E. coli (Seidler and Michel 1990, Meadows and Robinson 1991, Haehnel et al. 1994, Henry et al. 1997). Also, Mori and Cline (1998), and Wexler et al. (1998) reported that the signal

















A N H C
+ -- - ++ + - Precurss Delta pH AQKQDDVVDAVVSRRLALSVLIGAAAVGSKVSPADA OE23 Pathway - - ++ +
......ASAEGDAVAQAGRRAVIGLVATGIVGGALSQAARA OEI7
+ + - - - ++
...... MPVIKAORVVGDDVDGSNGRRSAMVFLAATLFSTAAVSASANA PSIN
- - ++ - ++
...... AQVESVQMSGERKTEGNNGRREMMFAAAAAAICSVAGVATA PSIIT

Sec +Pathway SSLKDFGVAIAVATAASIVLAGNAMA PC
+ -+- + - +
AFGLEHYGAKVTCSLQSDFKELAHKCVEASKIAGFALATSALVVSGASA OE33
++ - ++- +
...... APRSKIVCQQENDQQQPKKELAKVGANAAAALALSSVLLSSWSVAPDAAMA PSIF

Dual-Pakhway ++
MVSRRFGVIAVATAASIVLAGNAMA DT23, DT33


Figure 1-2. Lumen targeting domain (LTD) structure of precursors targeted to the cpSec, the Delta pH pathway, or to both pathways. The acidic (A), charged (N), hydrophobic (H) and cleavage (C) regions of LTD are shown. A hybrid dual targeting transit peptides (DT) is composed of the OE23 N region fused to the PC H region. The hydrophobic amino acids that comprise the H domain are underlined whereas charged amino acids are indicated with
(+) or (-). The sequences of the LTD for OE23, OE33 and PC correspond to the amino terminus determined by Bassham et al (1991). Precursors: PC (plastocyanin from Arabidopsis); OE17, OE23, OE33 (17, 23 and 33-kD proteins of the oxygen-evolving complex from maize, pea and pea); PSIN from Arabidopsis; PSIIT from cotton; PSIF from spinach.








peptide from an E. coli precursor could direct thylakoid passenger protein into the thylakoids.

The general structures of LTDs for both cpSec and Delta pH pathways are the same. With a compatible passenger, N, H and C domains in the LTD are sufficient for precursor targeting (Henry et al. 1994, 1997). The function of the A domain is currently unclear. Certain sub-domains of LTD determinate pathway specificity. For the Delta pH pathway, the N domain contains the specificity element. All the Delta pH pathway precursors contain a double arginine (RR) motif in the N domain, which is essential for the Delta pH pathway transport (Chaddock et al. 1995, Henry et al. 1997). It is likely that a receptor would interact with the double arginines, possibly combined with the non specific hydrophobic core, and bring the precursor to the Delta pH pathway translocation machinery. In contrast to the Delta pH pathway, the H domain requirement is more specific for cpSec pathway precursor targeting, while the N domain requirement is non specific (Henry et al. 1997). So, a different receptor could interact with the H domain and target the precursor to the cpSec pathway. The "domain determinant" hypothesis was further examined with artificial precursor DT-PC, which contains determinants for both pathways. DT-PC was constructed by replacing the N domain of cpSec pathway precursor PC with one from Delta pH pathway precursor OE23. This precursor incredibly can be efficiently transported on both pathways (Henry et al. 1997). Although "domain determinant elements" are key factors for pathway specificity, they alone seem insufficient for targeting. For example, amino acid residues surrounding the RR motif are important for the Delta pH pathway targeting (Bogsch et al. 1997).








In addition, results from Henry et al. (1994, 1997) and Clausmeyer et aL (1993) indicate that the mature protein plays a role in targeting and/or translocation on the cpSec pathway. For example, although a cpSec LTD could direct the Delta pH pathway passenger OE17 transport on the cpSec pathway, the transport was inefficient. Secondly, the hybrid (DT) transit peptide shown in Figure 1-2, while directing efficient transport of cpSec passengers on both pathways, was not able to direct efficient transport of the Delta pH passengers on the cpSec pathway.

In E. coli secretory proteins, a 30-residue-long region immediately downstream of the signal peptides has been termed the "export initiation domain". Introducing positively charged amino acid residues into the domain inhibits E. coli protein export at an early stage (Anderson & van Heijine 1991, 1993; Yoshihisa & Ito 1996). The inhibition could be reversed by adding negatively charged amino acids around the positively charged ones (Anderson & van Heijine 1991). Comparing the sequences of different precursors of the thylakoid cpSec pathway, we have observed that a 10-40 residue long region (adjacent to the LTD in PC and OE33, more distant from the LTD in PSIF) is characterized as having net zero or negative charge and containing a pair of special amino acid residues (NN or NQ, N-asparagine, Q-glutamine). This region may be analogous to the "export initiation domain". I speculate that it is this region that may be involved in the interaction between the precursor mature sequence and cpSec pathway transport machinery. The Delta pH pathway precursors generally have a net positive charge in this region and lack NN or NQ. This may be the reason why the Delta pH pathway passengers cannot efficiently access the cpSec pathway.








Studies have shown that the Delta pH pathway may be capable of transporting tightly folded precursors (Clark et al. 1997, Hynds et al. 1998). The natural precursor OE23 of the Delta pH pathway appears to be in a tightly folded conformation prior to transport (Creighton et al. 1995). Transport on the cpSec pathway appears require an unfolded precursor conformation (Ento et al. 1994, Hynds et al. 1998). So, it is also possible that Delta pH pathway precursor passengers cannot access the cpSec pathway because they have a tightly folded conformation.



Protein Transport on the epSec Pathway

The belief that chloroplasts were originated from a prokaryotic endosymbiont has driven speculation that thylakoid transport would be homologous to protein export from contemporary bacteria. The See system for protein export in E. coli has been investigated in detail by both biochemical and genetic techniques (Pugsley 1993, Rapoport et al. 1996). A preprotein translocase has been identified in E. coli (Brundage et al. 1990). The translocase consists of an integral membrane protein complex SecY/E/G and a peripheral membrane protein SecA. A working model has been proposed by Hartl et al. (1990). In this model, chaperone SecB, by binding to the precursor in the cytosol, prevents the precursor from aggregation. SecA binds the precursor-SecB complex and brings the complex to SecY/E/G. ATP binds to SecA and its hydrolysis by SecA facilitates translocation of the precursor. Besides ATP, a proton-motive force is stimulatory for the See system operation.

Some evidence suggests that SecA may function as a primary receptor for the precursor in bacterial protein transport. The direct interaction of SecA with the secretory








proteins was demonstrated by means of chemical cross-linking with EDAC (1-Ethyl-3[3-dimethlaminopropyl]-carbodiimide hydrochloride) and the interaction was signal peptide-dependent (Akita et al. 1990). A group of mutations in the SecA gene (termed prlD mutants) suppressed certain precursor malE signal peptide mutations. This suggested that SecA directly interact with the precursor signal sequence (Fikes and Bassford 1989; Puziss et al. 1989). SecA exists in both cytoplasm and membrane. It was suggested that cytosolic SecA does not function as the precursor receptor since, in reconstitution studies, an excess of SecA did not compete with SecY/E-bound SecA for limited amounts of added precursor-SecB complex (Hartl et al. 1990). Thus, it has been inferred that membrane association activates SecA for its receptor function. SecA is also known as the translocation ATPase of protein transport, so SecA could work as both an ATPase and a receptor.

There is also evidence to suggest that SecY is a precursor receptor. Antibodies against the N terminus of SecY are reported to prevent precursor binding to inverted membrane vesicles (Watanabe and Blobel 1989), as does trypsin treatment, which may inactivate SecY (Swidersky et al. 1990, 1992). Also, a group of mutations in the SecY gene (mutants prlA termed) suppress signal peptide mutations. By mapping the location of prlA mutations in SecY, Osborne and Silhavy (1993) hypothesized that the seventh transmembrane domain interacts with the hydrophobic core of the precursor signal peptide and plays a proofreading role. It is interesting that mutations in the SecE gene (prIG mutants) can also suppress signal peptides' mutations. So, it is possible that SecY or SecE may function as precursor receptors.









Thylakoid translocation of OE33 and PC has energy requirements similar to those of bacterial proteins that use the See system, implying that a Sec-like system is operational within chloroplast (Hulford et al. 1994, Yuan and Cline 1994a). Azide is known for its specific inhibition on SecA function in E. coli (Oliver et al. 1990). OE33 and PC transport is also inhibited by azide treatment, implying that a chloroplast homologue of SecA involved in the transport (Henry et al. 1994, Knott and Robinson 1994, Yuan and Cline 1994a). SecA- and SecY-homologous genes were found in the chloroplast genomes of several algae (Robinson and Klbsgen 1994) and subsequently the homologous genes were identified in plants (Berghofer et al. 1995, Nohara et al 1995, Laidler et al. 1995). The homologous genes in plants are nuclear encoded and named cpSecA and cpSecY. The requirement of cpSecA protein for OE33 and PC transport was clearly demonstrated in vitro with purified cpSecA from pea chloroplasts (Yuan et al 1994). Purified cpSecA replaced stromal extract and reconstituted transport of OE33 and plastocyanin with buffer- or urea-washed thylakoids in an azide-sensitive manner. Haward et al. (1997) observed a cpSecA-precursor crosslinking product during precursor binding. This at least indicates that the precursor is in direct contact with cpSecA. In vivo evidence for the cpSecA's role on the cpSec pathway protein transport came from the research with a maize mutant thai: the maize thai mutant was selectively inhibited in transport of OE33, plastocyanin and PSI-F (Voelker and Barkan 1995); the Thal gene was isolated and shown to encode maize cpSecA (Voelker et al. 1997).








Protein Transport on the Delta pH Pathway

The Delta pH pathway was first revealed by the discovery that two lumen proteins, OE17 and OE23, were transported into the thylakoids in such a unique manner that no soluble factor or NTPs were required for the transport. The ApH across the thylakoid membrane was the sole energy requirement (Cline et al. 1992). Little is known about the operating mechanism for the Delta pH pathway. It appears to work like a typical export system in E. coli or ER. First, precursors using the system have classical signal peptides (Cline and Henry 1996); second, the precursor transport uses loop topology and is likely translocated into the lumen from amino terminus to the carboxyl terminus (Fincher et al. 1998, Fincher and Cline unpublished). Although the Delta pH pathway does not require any soluble components, it certainly requires proteinaceous components on the thylakoid membrane. Transport cannot be conducted with protease pre-treated thylakoids (Robinson et al. 1996). Also, transport is saturated with high level of precursor and competed by unlabeled precursor (Cline et al. 1993). A component named Hcfl06 was identified as essential for Delta pH pathway function. Mutation in the Hcfl06 gene results in high-chlorophyll fluorescent (hc) phenotype and causes accumulation of Delta pH pathway intermediate precursors in the stroma (Voelker and Barkan 1995). Although the mutation diminishes the ability of the thylakoids to generate and maintain a transmembrane pH gradient, the ApH present was as high as in Thai, which transports precursor at normal rate on the Delta pH pathway. Isolated chloroplasts from hcfl06 mutant seedlings were totally unable to transport OE17 on the Delta pH pathway, but were still capable of transporting OE33 on the cpSec pathway (Settles et al. 1997). The Hcfl06 gene was cloned and the antibodies against Hefl 06 localized the protein on








thylakoid membranes. The bulk of the Hcfl06 protein is exposed to the stroma, suggesting that it may function as a receptor. Precursors on the Delta pH pathway have a critical RR motif in their lumen-targeting domain. If Hefl 06 functions as a receptor, it is likely that the RR is interacting with Hcfl 06 during the targeting process.

The Delta pH pathway was once considered to be eukaryotic innovation having evolved after endosymbiosis because all of its known substrates are absent from cyanobacteria, from which the chloroplast is believed to be evolved. However, database searches with the maize Hcfl 06 sequence revealed a homologous class of hypothetical bacterial proteins. This evidence strongly suggested that a similar pathway is operating in bacteria. The existence of the system in bacteria was also predicted by Berks (1996) based on the observation that a class of periplasmic proteins in bacteria are synthesized with signal peptides containing an RR motif. These redox proteins are apparently exported together with their redox cofactors and the exported proteins may be in a folded conformation. E. coli contains three homologues of Hcfl06 TatA, TatB and TatE, where tat stands for twin-arginine translocation. tatA and tatB genes are located in a four-gene operon, whereas the tatE is unlinked. tatA and tatE gene products have recently been shown by Sargent et al. (1998) to be required for the export of a range of proteins bearing the twin-arginine motif in their signal peptides. The Sec-system was shown to be unaffected in the mutant strains in which these genes were disrupted. Mutation of tatB also resulted in defective transport of RR-precursor (Weiner et al. 1998). Disruption of the tatC gene, which is also a member of the tat operon, results in a complete block in transport of five tested RR-precursor (Bogsch et al. 1998). TatC is a multispanning membrane protein and its homologous genes are present in a range of bacteria, plastids,









and mitochondria. Thus the Delta pH pathway is a novel pathway, discovered in plant thylakoids, but apparently exists in prokaryotes.



Summary and Perspective

Protein transport is required for the biogenesis of the chloroplast, the site of photosynthesis. Hundreds of chloroplast proteins are encoded in the cell nucleus and are synthesized in the cytosol. These proteins have to be transported into the chloroplast and functionally assembled.

A nuclear-encoded chloroplast protein is initially synthesized as a precursor protein that contains a transient transit peptide at its amino terminus. For a thylakoid lumen protein, its transit peptide consists of two parts: one is a stroma-targeting domain (STD) and another is a lumen-targeting domain (LTD).

Import of the precursor across the chloroplast envelope is governed by the STD of the transit peptide and accomplished by the cooperation of machinery components on both outer and inner envelope membranes. Such machinery is collectively called the general import apparatus. ATP hydrolysis is used to power import. The STD is cleaved by a processing protease in the stroma, resulting in either a mature stromal protein or an intermediate thylakoid protein precursor which is further directed to the thylakoids by the exposed LTD.

There appears to be one major pathway for protein import across the chloroplast envelope. However, there are two pathways for protein transport into the thylakoid lumen. One pathway is called the cpSec pathway since this pathway employs a chloroplast homologue of the bacterial SecA protein. A second pathway is named the








Delta pH pathway because in this pathway, the ApH across the thylakoid membrane is the only energy requirement for protein transport. Each pathway is specific for a subset of precursor proteins.

Protein transport into the thylakoid lumen is directed by the LTD. All precursors on the cpSec pathway or Delta pH pathway have a similar basic LTD structure containing three essential domains: N, H and C. Targeting to the cpSec pathway requires a specific hydrophobic H domain. Targeting to the Delta pH pathway requires a specific N domain (Henry et al 1997). A critical RR motif in the N domain is required for committing the precursor to the Delta pH pathway (Chaddock et al. 1995). The RR motif is thought to interact with a putative receptor on the thylakoid membrane. Further information is necessary to establish how the LTD is interacting with transport machinery and whether the precursor mature part is involved in the targeting step.

The cpSec pathway is powered by ATP hydrolysis and stimulated by ApH (Cline et al. 1992). The soluble component cpSecA is required for the transport (Yuan et al. 1994). Chloroplast homologues of bacterial SecY have been cloned (Laidler et al. 1995) and recent studies show that cpSecY is functional on the cpSec pathway (Hiroki et al. in preparation). Although Haward et al. (1997) demonstrated that precursor can form a complex with cpSecA on the thylakoid membrane, it was not clear from that study whether the cpSecA functions as a receptor or even if the interaction between precursor and cpSecA is specific. Also it is not known whether the interaction involves cpSecY. Further work is required to show how cpSecA engages precursors in targeting and translocation.








The ApH is the only energy requirement for the Delta pH pathway transport and no soluble component is required (Cline et al. 1992). The thylakoid protein Hcfl06 was recently identified as a component required for the pathway (Settles et al. 1997). Although the Delta pathway was first discovered by a biochemical approach, the machinery components were identified by genetic methods. However, development of new biochemical approaches is necessary to delineate the role of identified components in the translocation mechanism.

In the studies presented in this dissertation, I have conceptually and experimentally divided protein transport across the thylakoid membrane into two steps. The first is targeting in which the precursor is committed to the pathway. The second is translocation, wherein the protein actually is moved by the transport machinery across the membrane. I hypothesize that targeting results from a specific interaction between the precursor and components of the protein transport machinery. Each protein transport pathway has its own components to recognize the precursor and such components are referred as precursor receptors. So far, protein transport machinery components (receptors) related to pathway selection have not been identified.

The purpose of this study is to examine the molecular determinants and steps of the process that result in precursor commitment on the cpSec pathway or the Delta pH pathway. The specific questions addressed are: 1. Is there an earliest detectable precursor targeting step that commits the precursor on the cpSec pathway or the Delta pH pathway? Specifically, is there a detectable complex formed during the precursor targeting step? 2. Are there any receptors that interact specifically with the precursor during the precursor





21


targeting step? 3. What are the elements of precursors necessary for targeting? 4. What are the energy requirements for precursor targeting and translocation?

The long range goal is to determine underlying reasons for the existence of multiple pathways for protein transport into the thylakoids. To achieve the goal, it will first be necessary to define each pathway. The successful completion of this study will provide further understanding of thylakoid protein transport mechanisms.













CHAPTER 2
PROTEIN TARGETING AND TRANSLOCATION ON THE cpSEC PATHWAY Abstract

To investigate the basis for pathway-specific targeting, the characteristics of membrane binding and chase of precursors on the cpSec pathway were examined. Previous studies showed that the epSec pathway precursor iOE33 could form a productive intermediate and be crosslinked to cpSecA on the thylakoid membrane (Haward et al. 1997). Our results support the notion that precursor commitment to the thylakoid cpSec pathway occurs on the membrane, rather than in the soluble stroma. Precursor binding to the thylakoid membrane required stromal protein, of which cpSecA was the essential component, and occurred when transport was inhibited by removing ATP from the assay with apyrase. Sodium azide and ionophores, although inhibiting transport, did not result in increased precursor binding. Subsequent transport of bound precursor required ATP, but was not stimulated by additional cpSecA. Upon interaction with thylakoids, precursors could be crosslinked into a high molecular weight complex (cpSec complex) that migrated anomolously in SDS-PAGE. The crosslinked product was immunoprecipitated with antibodies to cpSecA as well as with antibodies to cpSecY. Formation of the cpSec complex was strictly precursor-specific. Precursors capable of being transported by the cpSec pathway were crosslinked into the complex, whereas precursors transported by the Delta pH pathway and SRP pathway were not. Of interest is the fact that precursors consisting of a cpSec-compatible signal peptide fused to a Delta








pH passenger protein produced a higher molecular weight crosslinking product that was not immunoprecipitated by antibodies to cpSecA. Previous studies have shown that Delta pH passenger proteins are incapable of efficient transport on the cpSec pathway. This later result suggests that commitment to the cpSec pathway at an early step involves not only the signal peptide, but elements of the passenger protein as well.



Introduction

Nucleus-encoded thylakoid lumen-resident proteins are synthesized in the cytosol as precursors with bipartite amino-terminal transit peptides. Precursors are localized to the lumen by a two-step mechanism (Cline and Henry 1996, Schnell 1998, Robinson et al. 1998). In the first step, the precursors are imported across the chloroplast envelope into the stroma. This step is governed by the stromal targeting domain of the transit peptide. A stromal processing protease removes the stromal targeting domain, thereby producing a stromal intermediate precursor, which is transported across the thylakoid membrane in the second step. Thylakoid transport is directed by the lumen-targeting domain of the transit peptide, which is removed by the thylakoid processing protease.

The cpSec pathway is one of two pathways for protein transport into thylakoid lumen. OE33, PC and PSIF (F sub-unit of Photosystem I) are three proteins known to use the cpSec pathway for their transport. Protein transport on the cpSec pathway requires ATP, a stromal protein cpSecA (Yuan et al. 1994), and a membrane protein cpSecY (Mori et al., in preparation). According to Haward et al. (1997), when ATP was eliminated with apyrase, iOE33 transport was inhibited, resulting in accumulation of bound precursor on the thylakoid membrane. The bound precursor could be chased into








thylakoid lumen upon ATP addition. Precursor binding is considered to be a targeting step whereas the chase is the translocation step. cpSecA and cpSecY are homologues of the bacterial SecA and SecY proteins (Berghofer et al. 1995, Nohara et al. 1995, Laidler et al. 1995). The bacterial SecA protein is a translocation ATPase that binds to precursors and SecY and "pushes" peptide segments across the membrane through a cycle of insertion and deinsertion. SecY is a membrane protein that is thought to play a major role in forming the protein translocation channel with SecE component (Akiyama and Ito 1985, Meyer et al. 1999).

Committing a precursor protein to a specific pathway, i.e. targeting, is believed to be initiated by an interaction between precursor and a receptor. It has been shown that the lumen-targeting domain determines whether a protein is transported on the cpSec or Delta pH pathway (Henry et al. 1994). It has also been shown that the mature protein domain, i.e. the passenger, can greatly affect the efficiency of transport by the cpSec pathway. For example, although a cpSec LTD directed transport of OE17 on the cpSec pathway, the transport was inefficient. Secondly, the hybrid (DT) transit peptide, while directing efficient transport of Sec passengers on both pathways, was only able to direct efficient transport of Delta pH passengers on the Delta pH pathway (Henry et al. 1994, 1997). Haward et al. (1997) reported that iOE33 formed a complex on the thylakoid membrane with cpSecA and suggested that cpSecA may function as a receptor. However, it was not known which part of the precursor bound to cpSecA and more information is required to determine whether cpSecA functions as a receptor.

I am interested in learning how the cpSec pathway system recognizes its respective substrates and commits them to the pathway. I have attempted to describe and








characterize the first committed step on the cpSec pathway and to define in general terms the sequences required for commitment. My results show that the first observable interaction of precursors with the cpSec pathway occurs on the membrane and results in formation of a large complex that includes precursor, cpSecA, and cpSecY. This interaction requires a cpSec LTD and additionally a cpSec-compatible flanking region. Proteins that possess dual targeting signal peptides fused to Delta pH passenger proteins are capable of binding to the membranes as well as chasing into the lumen on the delta pH pathway. However, these proteins do not form a complex with cpSecA.



Materials and Methods

Preparation of precursors

In vitro transcription plasmids for iOE33, iOE23 and LHCP were described as Cline et al. (1993). Preparation of tPC, DT33, DT17 and DT23 were described as Henry et al. (1997).

Preparation of Chloroplasts, Lysates, Thylakoids and Stroma

Chloroplasts were isolated from 9 to 10-day-old seedlings of pea (Laxton's Progress 9). Lysates, thylakoids and stromal extract were prepared from the intact chloroplasts (Cline et al. 1993)

Preparation of Purified cpSecA

CpSecA was purified from stromal extract as described by Yuan et al. (1994b), except that studies reported here used cpSecA obtained after the Mono-Q ion exchange step. The concentration of purified cpSecA was estimated by Coomassie staining of SDS polyacrylamide gels using BSA as a standard.








Thylakoid Protein Transport Assay

Transport assays were conducted essentially as described previously (Cline et al. 1993): 25 jLg chlorophyll of thylakoids or lysates in 25 pl of import buffer (50 mM Hepes buffer pH 8, 0.33 M sorbitol) was mixed with 30 pl HKM buffer (10 mM Hepes buffer pH 8, 10 mM MgC12) with or without 300 gg of stromal protein, and incubated with 20 jpl 1 fold diluted in vitro translated precursor in import buffer with 30 mM leucine. ATP or apyrase, ionophores, or azide were added in respective assays. The total volume of each assay was about 80 pl. Assays were incubated under 70 E mf'2s"' white light (or dark) for 15-30 minutes at 25*C. After incubation, thylakoids were recovered by centrifugation and were washed once with import buffer. One half of each sample of recovered membranes was analyzed directly on SDS-PAGE. The other half was treated with 10 pg thermolysin in 100 pl of import buffer (Cline 1986) and then analyzed by SDS-PAGE and fluorography.

Precursor Binding and Chase Assays

Thylakoids or chloroplast lysate (in 25 pl import buffer) equivalent to 25 pg chlorophyll was mixed with 40 gl HKM buffer with or without 400 jg of stromal protein. Two jig cpSecA, ionophores, or azide were added to respective assays. The mixture was incubated for 10 minutes in the dark in the presence of 2 il (units) apyrase. About 10 pl in vitro translated precursor was added and the incubation continued for 15 minute at 25�C in the dark. Following the incubation, thylakoid membranes were recovered by centrifugation, washed twice with import buffer, and divided into two equal portions in fresh microfuge tubes. One portion was analyzed directly. The other portion was incubated under chase conditions. The chase was conducted under 70 iE m-2s white








light for 15-30 minutes at 25�C in a total of 37.5 p l of import buffer containing -200 pg protein of stromal extract and ImM DTT. Where designated, 12.5 pg chlorophyll aliquots of thylakoids recovered from binding or chase samples were treated with 10 pg thermolysin in 100 p l of import buffer (Cline 1986). Preparation of Urea-washed or Thermolysin Treated Thylakoids for Binding Assays and Urea or Na2CO3 Wash of the Thylakoid Bound Precursor

Urea-washed thylakoids were prepared by incubating 0.5 mg chlorophyll of thylakoids in 1 ml of 2 M urea in import buffer for 8-10 minutes on ice. Thylakoids were recovered by centrifugation and washed once with import buffer. Thermolysin-treated thylakoids were prepared by incubating 0.33 mg chlorophyll of thylakoids in 1 ml import buffer containing 100 pg thermolysin on ice for 40 minutes. Thylakoids were recovered by centrifugation and washed twice with import buffer containing 10 mM EDTA and once with import buffer. Urea or Na2CO3 wash of the thylakoids recovered from binding assays was accomplished by incubating 0.5 mg chlorophyll/ml of thylakoids with 6 M urea or 0.1 M Na2CO3 (in water) on ice for 30 minutes. The thylakoids were recovered by centrifugation (at 3200x G for 8 minutes) and washed twice with import buffer. Cross-linking Reactions

Binding assay mixtures, following incubation for 15 minute, received 0-2 mM crosslinker from 0-40mM stocks in DMSO (for DSS (disuccinimidyl suberate), MBS (mMaleimidobenzoyl-N-hydroxysuccinimide ester) and APDP ( N-[4-(pAzidosalicylamido) butyl]-3'-[2'-pyridyldithio] propionamide) ) or H20 (for BS3 (bis [sulfosuccinimidyl] suberate)). Cross-linking was allowed to proceed at room temperature for 30 min or at 0OC for 2 hours and was terminated with 50 mM Tris-HC1








pH 7.5 from a 1 M stock. After the cross-linking reaction, the membrane fraction was recovered by centrifugation and washed with import buffer. Preparation of Antibodies

LHCP and CpSecA antibodies were prepared as described by Payan and Cline (1991) and Yuan et al. (1994b), respectively. Peptides NH2CKLQDLQKKEGEAGRKK-COOH and NH2-CDDVSEQLKRQGASIPLVRPGKCOOIH, termed Inl and In2 respectively, correspond to two internal stromal-facing regions of cpSecY. Inl, In2 and the antibodies against the peptides were prepared by BioSynthesis company (TX). InI and In2 were conjugated via their N-terminals to carrier keyhole limpet hemocyanin with crosslinker MBS and the crosslinked products were used as antigens. The peptide NH2-CRAEIISQKYNIELYDFDKY-COOH (termed Cterm), corresponding to the C-terminal stromal-facing region of pea cpSecY, was made and cross-linked by Genosys Biotechnologies company (Woodlads, TX). The antibodies raised against the Keyhole limpet hemocyanin-linked C-term peptide were prepared by Cocolico Company and described by Mori et al. (In preparation). Immunoprecipitation of the Cross-linked Complex

Twenty-five gg chlorophyll of thylakoid membrane recovered from each crosslinking assay was resuspended in 75 gl of 10 mM HEPES/KOH (pH 8), 10 mM MgCl2, followed by 4 Il 20% SDS. After 30 minute incubation at 37.C, the sample was brought to 800 il with 10 mM HEPES/KOH (pH 8), 10 mM MgC2, 0.2% Triton X-100. Ten to fifteen pl serum, with or without 10-20 jig synthetic peptides, was added and the sample was mixed end-over-end at 4oC overnight. Forty pil 1:1 protein A Sepharose slurry in 10 mM HEPES/KOH (pH 8), 10 mM MgCl2 was added and the sample was mixed end-








over-end at 4C for 1 hour. The Sepharose beads were pelleted by centrifugation at 500x G for 3 minutes, resuspended in 10 mM HEPES/KOH (pH 8), 10 mM MgCl2, transferred to a new tube, pelleted, and then treated with 20 jl 2X SDS sample buffer. Analysis of Samples

Samples recovered from the assays were subjected to SDS-PAGE and fluorography. Most samples were analyzed with 12.5% acrylamide concentration gels. Cross-linking and immunoprecipitation samples were analyzed with 7.5% gels. Quantification of the amount of radiolabeled proteins resulting from these assays was accomplished by scintillation counting of radiolabeled proteins extracted from excised gel bands (Cline 1986).



Results

No Interaction can be Detected between cpSec Pathway Precursor and Soluble epSecA

CpSecA is the only soluble chloroplast component required for protein transport on the cpSec pathway and, therefore, the only candidate for a specific interaction in the soluble fraction. Bacterial SecA is reported to interact weakly with precursor in solution (Hartl et aL 1990) and can be crosslinked to precursors with EDAC (1-Ethyl-3-[3dimethlaminopropyl]-carbodiimide hydrochloride) (Akita et al. 1990). In preliminary studies, we were unable to detect any interactions between cpSec pathway precursors and cpSecA in solution using several different interaction assays. These included nondenaturing PAGE (gel shift assays), gel filtration of the authentic stromal intermediate








isolated from chloroplasts, crosslinking combined with immunoprecipitation, and nascent chain crosslinking combined with imnmunoprecipitation (data not shown). iOE33 Binds to the Thylakoid Membrane when Transport is Prevented by Eliminating the ATP

In E. coli, strong binding to Sec components occurs on the membrane (Hartd et al. 1990). In addition, Haward et al. (1997) reported that the precursor iOE33 binds productively to thylakoids and can be crosslinked to cpSecA. Here we have further characterized this binding and investigated the possibility that membrane binding represents the first committed step on the cpSec pathway. [3 HI-labeled precursor was incubated with thylakoids under assay conditions either conducive for transport or where a single transport requirement was withheld (Figure 2-1). Wheat iOE33 was used as the precursor as it is the most efficient cpSec pathway substrate under our conditions. As in previous studies (Yuan et al. 1994a, Hulford et al. 1994), iOE33 was transported into the thylakoid lumen when stromal extract, ATP and ApH were present, as judged by processing to the mature size and inaccessibility to exogenous protease. Transport was inhibited when assays were conducted in the presence of ionophores that dissipate ApH, or azide, a SecA inhibitor, or in the absence of ATP (apyrase was used to scavenge all traces of ATP). In the absence of ATP/presence of apyrase, precursor binding to the thylakoids membrane was stimulated (Figure 2-1, lane 1 and lane 4). The amount of bound precursor was - 2.6 fold higher in the presence of apyrase than that under any of the other conditions. In the presence of apyrase, about 2.6% of the added precursor in the transport mixture was bound on the thylakoid membrane. Of interest is that, although








light - + + + + + ATP - + + - + +
nig/val - - + - - SE + + + + - +
apyrase + - - + -
azide - - - - - +
P 1 2 3 4 5 6
-iOE33
'mOE33


--mOE33



Figure 2-1. cpSec-pathway transport intermediate accumulated on the thylakoid membrane when transport was arrested with apyrase. Transport assays with [3H]-leucine labeled iOE33 precursor were conducted with isolated thylakoids under conditions (shown above the panel) designed to test the effects of removing ATP or stromal extract (SE), of dissipating the ApH, or of inhibiting cpSecA with azide. Assays (751W) contained 412.5 lg protein of stromal extract (SE), 5 mM Mg-ATP, nigericin and valinomycin (nig/val) at 0.5 jiM and 1 pM, respectively, and 10 mM sodium azide as indicated above the panel Apyrase treatment was 2 unit per 75 l.d assay. For the top panel, the thylakoid membranes were recovered by centrifugation and washed with import buffer. Membranes in the lower panel were treated with the protease thermolysin prior to washing. Samples were then analyzed with SDS-PAGE and fluorography. Each lane contained 12.5 ig chlorophyll (100%) of thylakoid membranes present in each assay. Lane P represents 0.25 pl (5%) precursor added to the assay reaction. The positions of the precursor iOE33 and the mature form (mOE33) are marked.








ionophores and azide inhibited transport of iOE33, they did not result in increased binding.



Binding of iOE33 to Thylakoids is Productive and Stimulated by epSecA

To determine if precursor was productively bound in the absence of ATP/presence of apyrase, membranes, washed following a binding assay, were incubated with stromal extract and ATP at 25�C for 30 minutes (Figure 2-2). Approximately 11% of the bound precursor was subsequently transported (chased) into the lumen and processed to mature size, whereas precursor bound without stromal extract or cpSecA failed to be chased into the lumen (Figure 2-2A). cpSecA is the required stromal component for iOE33 transport (Yuan et al. 1994b). Purified cpSecA significantly increased precursor binding and the subsequent chase over that of buffer alone. The amount of cpSecA added is approximately equivalent to the amount in unfractionated stromal extract. The relatively low percentage of precursor that is chased under our conditions may reflect the fact that transport competence ofthylakoids is significantly reduced by the washing steps. This is shown in Figure 2-2B. A transport assay conducted with apyrase treated and bufferwashed thylakoids plus stromal extract yielded -56% transport compared to freshly prepared membranes (compare lane 1 to lane 4).

It was shown in Figure 2-1 that azide or ionophores alone, while inhibiting transport, did not result in increased precursor binding. Figure 2-3 examines the effects of azide and the ApH on binding in the absence of ATP/presence of apyrase. Azide slightly decreased, whereas ionophores had no significant effect on binding. Precursor bound under all those conditions was chased into the lumen, although with varying efficiencies. Of interest is










A B

Binding Binding Chase Transport
SE HKM A SE HKM A SEHKM A SE HKM A TC
P1 2 3 4 5 6 7 8 9 1 2 3 4





-PM/Bn cewsand CPMIBand 1
CPM/Band s78os0
S318 se


1 2 3 7 8 9 1 2 3 4
Binding Chase Transport



Figure 2-2. cpSecA stimulates iOE33 binding on the thylakoid membrane and the binding is productive. Panel A: 15 p l [3H]-labeled iOE33 precursor in vitro translation product was incubated with 37.5 pg chlorophyll of thylakoids (in 37.5 pl import buffer) in the presence of 3 pl (equal to 3 units) apyrase in import buffer for 15 minute at 25oC in a total 115.5 pl binding assay. Binding assays received -800 pg of stromal protein in 60 pl 10 mM Hepes/KOH pH 8, 10 mM MgCl2 (HKM buffer) (lanes 1, 4, 7), or 60 p1 HKM alone (lanes 2, 5, 8), or -3 pg purified cpSecA in 60 pI1 HKM buffer (lanes 3, 6, 9). After binding incubation, the thylakoid membranes were recovered, washed twice with import buffer, divided into three equal aliquots and transferred to new tubes. One aliquot was directly analyzed (lanes 1-3). A second aliquot was treated with thermolysin (lanes 4-6). The third aliquot was incubated under chase conditions and then treated with thermolysin (lane 7-9). Each lane contains 100% of thylakoid membrane from each assay. Lane p represents about 0.25 gl precursor in vitro translation product. Panel B: Thylakoid transport assays conducted with fresh thylakoids (Lanes 1, 2, 3) or thylakoids incubated with apyrase under conditions of a binding assay and then washed twice with import buffer before use (lane 4). All reactions contained 5 mM ATP. Reactions of lanes 1, 4 contain stromal extract; lane 2 buffer; lane 3 purified cpSecA. Final samples were treated with thermolysin before being analyzed by SDS-PAGE and fluorography. The charts display radioactivity (CPM) of corresponding bands.












Binding Binding Chase
1 2 3 4 1 2 3 4 1 234


Protease
post-treatment

Figure 2-3. The effects of ApH and azide to iOE33 binding to the thylakoid membrane. iOE33 binding assays were conducted in the presence of stroma and apyrase. Binding assays in lane 2, 3 and 4 were supplemented with 3 jiM CCCP (Carbonylcyanide 3chlorophenylhydrazone), 0.5 pM/1 jpM nig/val or 10 mM sodium azide respectively. Chase assays were all conducted under identical condition (see Methods).








that precursor bound in the presence of ionophores was chased into the lumen with efficiency greater than precursor bound under other conditions. Because ionophore effects of nig/val persist through washing steps, this was the first indication that a ApH was not required for the chase reaction.


Soluble cpSecA is not Required for the Chase of Bound Precursor

The requirements for chase of bound precursor into the thylakoid lumen were investigated (Figure 2-4). Maximum chase of bound iOE33 resulted when stromal extract was included in the re-incubation mixture. ATP alone was able to partially stimulate chase, but chase with ATP plus stromal extract was no greater than stromal extract alone. Purified cpSecA was ineffective in stimulating chase, indicating that cpSecA is committed at the early binding step and that free cpSecA is largely ineffective at the chase stage. This is consistent with observations in the bacterial Sec system that excess soluble SecA did not compete with membrane-bound SecA for limited amounts of added precursor-SecB complex (Hartl et al. 1990) and that bacterial SecA does not cycle off the membrane during protein transport (Chen et al. 1996). Although free cpSecA was not required for the chase, bound cpSecA played an important role during the chase since azide partially inhibited the chase (Figure 2-4B). Unexpectedly, the chase was stimulated by ionophores. Yet, the thylakoids' ability to generate a ApH was not likely destroyed by manipulations involved in binding and washing because apyrase treated and washed thylakoids were still capable of transporting fresh precursor in an ionophore-sensitive fashion (data not shown). Rather, the energy required for transport was derived from ATP because addition of apyrase prevented chase of precursor into the lumen (data not















49'
Chase 4 Chase
1 2 3 4 5 67 B 2 3 4
A B
-JO -* iW 4



Chase .c
Condition < z < +
ATP+SE
<



Figure 2-4. Requirements for chase of bound precursor into the lumen. A. 28 Al [3H]leucine labeled iOE33 precursor was incubated with 87.5 Ag chlorophyll of chloroplast lysate (in 110 Al import buffer) and -1200 Ag protein of stromal extract (in 120 jpl HKM buffer) in the presence of 7 units (ptl) apyrase for 15 minute at 25(2. Thylakoid membranes were then recovered and washed twice with import buffer and divided into seven equal portions. Each portion was transferred to a new tube. One portion was analyzed directly (lane 1). The remaining portions were further assayed for chase under different conditions. Assays contained import buffer (lanes 2, 4), 200 jig stromal protein
(SE)(lanes 3, 5), -0.8 pjig purified cpSecA (lanes 6, 7) were incubated in the presence (lanes 4, 5, 7) or absence (lanes 2, 3, 6) of 5 mM ATP for 30 min. at 25C in light in a total 37.5 Al. Recovered thylakoids were treated with the thermolysin and then analyzed by SDS-PAGE and fluorography. B. The binding incubation was similar to that shown in panel A. During the chase step, 12.5 ig chlorophyll of thylakoid membrane with bound precursor were resuspend in buffer containing 200lg protein of stroma extract and 5 mM ATP plus 1 pM/0.5 pM nigericin/valinomycin (N/V) or 10 mM sodium azide (Azide) or import buffer (IB).









shown). Stromal extract contains small amounts of ATP and presumably this was sufficient to power the transport reaction in assays containing stromal extract alone. Precursor Binding to Thylakoids is Mediated by Protein-protein Interactions

Further investigation into the nature of the observed precursor binding is shown in Figure 2-5. Binding was maximal with membranes that were either buffer- or ureawashed and when stromal extract was present in the reaction mixture. Approximately 2.12.7 % of the added precursor was bound to the membrane under these conditions. Binding to protease-treated membranes was greatly reduced (only ~-.4% of the added precursor was bound to the membrane). Following the binding reaction, thylakoids were washed with 0.1 M Na2CO3 or 6 M urea to assess the nature and strength of the binding. The majority of precursor bound to buffer- or urea- prewashed membranes (-60%) was removed by the Na2CO3 and urea washes, whereas the low level of precursor bound to protease-treated membranes was largely resistant. This, combined with previous observations that transport is eliminated by protease (Robinson et al. 1996), but not ureatreatment of thylakoids (unpublished data) suggests that iOE33 binding involves proteinprotein interaction, whereas a low level of precursor can non-specifically interact with the thylakoid surface.



Productive Binding Results in Formation of a Complex on Thylakoids

The above results imply that binding of iOE33 involves cpSecA and thylakoid protein(s), and suggests the formation of a complex on the membrane, similar to the Sec complex shown for bacterial membranes (Brundage et al. 1990). To assess whether such a complex exists, a binding reaction was conducted for 15 minutes, after which the cross-













Treatment s to the thylakoids before the binding


IB wash (lane 1-6)
1 2 3 4 5 SE + + +- -


Bound iOE33 l -


Urea wash (lane 7-12)
6 7 8 9 10 11 12 13
-+ + + - - +



415 55a- -


Protease incubation
(lane 13-18)
14 15 16 17 18 + + - .


IB
Thylakoids wash NB after the binding Na2CO3
Urea


10000
8000 6000
4000 2000
0


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18


Figure 2-5. iOE33 precursor binding to the thylakoid membrane is mediated by proteinprotein interactions. Nine ll [3H] labeled iOE33 translation product was incubated with 37.5 gg chlorophyll of import buffer-washed thylakoids or 2 M urea-washed thylakoids or thermolysin (100 gg/ml)- treated thylakoids in the presence or absence of -525 jig stromal proteins in a total 112.5 pl. All assays contained 3 unit of apyrase and were incubated in the dark at room temperature for ~30 min. After the incubation, each sample was divided into three equal portions. The thylakoid membranes in each portion were washed with import buffer (lanes 1, 4, 7, 10, 13, 16) or 0.1 M sodium carbonate (lanes 2, 5, 8, 11, 14, 17) or 6 M urea (lanes 3, 6, 9, 12, 15, 18). Thylakoid membranes recovered from the assays (12.5 jig chlorophyll) by centrifugation were analyzed by SDS-PAGE and fluorography.








linking agent DSS (disuccinimidyl suberate) was added to stabilize any possible complex. Multiple cross-linking products, apparent as higher molecular weight radioactive bands, were present in both the soluble, stromal fraction and the thylakoid membrane fraction (Figure 2-6 A, (1) and (2)). Because of the requirement for cpSecA in the binding reaction, crosslinked products were subjected to immunoprecipitation with antibodies to cpSecA. Soluble crosslinking products were not immunoprecipitated (Figure 2-6B). However, the large crosslinking product that migrates near the top of the separating gel was immunoprecipitated from the membrane fraction with antibodies to cpSecA (Figure 2-6A, (3)) but not with preimmune serum (data not shown, but see Figure 2-7). Further experiments (Figure 2-6B) indicated that the formation of this crosslinked product required both stromal extract and the thylakoid membrane and that purified cpSecA could replace stromal extract in this reaction. From these data, it can be concluded that iOE33 forms a complex with other proteins on the thylakoid membrane and that cpSecA is part of the complex.

In a standard binding assay containing thylakoids and stromal extract, ~2.4% of the added precursor remained bound to the recovered and washed thylakoid membrane. When 1 mM DSS was added at the end of the binding reaction, -3.5% of the added precursor was recovered as the cross-linked complex. Over 50% of the cross-linked complex was immunoprecipitated with antibody against cpSecA. When thylakoids recovered from a binding assay were treated with DSS following the washing steps, the same cross-linked complex was formed, though a lesser amount than when crosslinker was added directly to the binding reaction (data not shown). When the binding assays were conducted in the presence of ionophores or sodium azide alone, cpSecA-containing









(1) (2) (3)
A
!!



DSS(mM) 0 0.10.5 1 2 0 0.1 0.5 1 2 0 0.10.5 1 2 SE ......L. I�m S
S M SM SM
B






Figure 2-6. Formation of a complex between precursor and components of the cpSec machinery. A. Crosslinkers stabilize a large cpSecA-containing complex on the membrane. Ten 1.l [3H] labeled iOE33 in vitro translation product was incubated with 25 gg chlorophyll of chloroplast lysate plus 40 pl (400 lkg of protein) additional stromal extract in the presence of 2 units (in Il l import buffer ) apyrase. After 15 minutes incubation, 4 gl of varying amounts of DSS in DMSO were added to the reaction mixture, which was further incubated for 2 hours in an ice bath. The final DSS cocentrations in the mixture were 0-2 mM. The cross-linking reaction was terminated by adding 4 pdl I M Tris-HC1, pH 7.5. The membrane and stromal fractions were separated by centrifugation and aliquots of the samples were further analyzed by anti-cpSecA immunoprecipitation. Final samples were analyzed by SDS-PAGE (7.5% gels) and fluorography. Panels: (1), the cross-linking products in 7.5 pil supernatant from crosslinking mixture (2), the cross-linking products on the 6.25 jig chlorophyll of membrane;
(3), the antiSecA immunoprecipitation products of the cross-linking products on the about 12.5 ttg chlorophyll of membrane. B. cpSecA is required for complex formation. Ten tl [3H]-labeled iOE33 precursor was incubated either with stromal extract (SE, -600 jig protein ) or with 25 jtg chlorophyll of thylakoids (T) or with the thylakoids plus purified cpSecA (T+SecA, 1.6 pg cpSecA) or with the thylakoids plus stroma extract (T+SE) and the reactions were crosslinked with 1 mM DSS. The reactions were then subjected to immunoprecipitation with antibody to cpSecA. Immunoprecipitation products from the soluble fraction (S) or the membrane (M) are shown.








crosslinking products were not obtained (data not shown), indicating a strong correlation between productive binding and the ability to form a crosslinked complex. cpSecY is Part of the cpSec Complex

In bacteria, the interaction of precursor and SecA with the membrane results in a complex with the SecY/E/G protein. To determine if the precursor-cpSecA complex also contains cpSecY, immunoprecipitations were conducted with antibodies generated to cpSecY. Antibodies were raised to synthetic peptides that correspond to several regions of the predicted pea cpSecY sequence (see Methods). Initially, antibodies were raised to peptides corresponding to two internal stromal-facing regions of cpSecY. These antibodies are capable of specifically immunoprecipitating the cpSecY translation product but fatil to give a band on immunoblots (Mori and Cline, unpublished). Subsequently, a third antibody was raised to the extreme carboxyl terminus of cpSecY. This antibody immunodecorates a 42 kD pea thylakoid protein, presumed the cpSecY, located in the stroma lamellae (Mori et al., in preparation). All three antibodies immunoprecipitated the large precursor cpSecA complex (Figure 2-7 A and B). The complex was not precipitated with pre-immune serum or when immunoprecipitation was conducted in the presence of the corresponding synthetic peptides. To verify that DSS cross-linker was not simply crosslinking the bound precursor to any component on the thylakoid membrane, immunoprecipitations were conducted with antibodies against LHCP, a major protein of the thylakoid membrane but not related to the cpSec pathway. No radiolabeled cross-linking products were immunoprecipitated with these antibodies (Figure 2-7C).












antiSecY Sern antSecA aatHCP
antiLIICP
Preln2 Inl In2 Pie Im
- T 3 4 5 6 7XSI XMI 123456 7
A W B


cpSecYsynthetic + + + +
peptide XP antiSecY C-term antiSecA
XP Im Im Pre Im Im
1 2 3 4 5 6
C War,


cpSecYsynthetic . + . . +
peptide


Figure 2-7. cpSecY is part of the cpSec complex. The experimental conditions were similar to Figure 2-6. Cross-linking reactions were conducted with I mM DSS. A. Crosslinking products on 25 gg chlorophyll of thylakoid membranes were immunoprecipitated with antibodies against two internal stroma-facing regions of cpSecY (Inl, In2 synthetic peptides) in the presence or absence of the corresponded synthetic peptides (1-2ug/ul serum). AntiSecA immunoprecipitation in the presence of SecY-synthetic-peptide (In2) was used as controL The preimmue sera for both anticpSecY (anti-In2) and antiSecA (Lane 1 and Lane 2 respectively) were also used as controls. B. iOE33 precursor crosslinking products from both the stroma (XSI) and the thylakoid membrane(XMI) were immunoprecipitated with antiLHCP serum. C. Cross-linking products on the 25 pg chlorophyll of thylakoid membrane were immunoprecipitated with antibodies against the extreme carboxyl terminus of cpSecY in the presence or absence of the cpSecY synthetic peptide. Anti cpSecA immunoprecipitations in the presence or absence of cpSecY synthetic peptide demonstrated the specificity of the peptide effect. The preimmue sera of cpSecY antibody (Pre) was also used as control. Lane 1 contains cross-linking products of 12.5 jig chlorophyll of thylakoid membrane. The remaining lanes contain immunoprecipitate obtained from 25 jg chlorophyll of crosslinked thylakoids.








Since the cpSec complex contained cpSecY, we further wanted to know if cpSecY played any role in the precursor binding. Antibodies against the C-terminal region of cpSecY inhibited protein transport on the cpSec pathway (Mori et al., in preparation). After thylakoids were pre-incubated with these antibodies, precursor binding on the thylakoid membranes were significantly reduced. The corresponding antigen reversed the precursor binding reduction (Figure 2-8).



Formation of the epSecA-containing Complex is Pathway-specific

It has been shown that precursor proteins transported by the Delta pH pathway and cpSRP pathways do not require cpSecA (Yuan et al. 1994). Therefore these precursors are not expected to form a cpSecA containing complex. Similar crosslinking reactions were conducted with precursors from different pathways (Figure 2-9, Panel A). Several different crosslinking agents were used to ensure that a crosslinking product would be obtained if precursors were in contact with cpSecA. CpSecA-containing complexes were obtained with iOE33 and tPC, both are substrates of the cpSec pathway. In contrast, no cpSecA-containing complexes were obtained with the Delta pH pathway substrate iOE23 or the cpSRP pathway substrate LHCP. However, very little crosslinking was achieved for iOE23, possibly because it does not bind thylakoids tightly. Additional experiments were conducted with Delta pH pathway precursors that do bind to the membrane (Figure 2-9, Panel B). tOE17 is a truncated form of iOE17; DT-17 and DT-23 are precursors that possess a dual targeting (DT) signal peptide. When the DT-signal peptide is fused to a cpSec pathway passenger protein, the resulting precursors are transported on both the cpSec and the Delta pH pathway. However, fusion proteins containing Delta pH




















Buf Im PI Im Buf
TP 1 2 3 4 5



Antigen - - - + +


Figure 2-8. iOE33 binding was reduced on anti-cpSecY pre-treated thylakoids. 0.33 jig chlorophyll /jl of thylakoids were pre-incubated with 0.8 jtg IgG /l of anti-cpSecY (Cterm.) or preimmune in 3% BSA-IBM buffer in the presence or absence of 15 pM cpSecY (C-term.) synthetic peptide. The incubations were for one hour in an ice bath. Thylakoid membranes were washed once with IB buffer and used for iOE33 binding assays as describing in methods. Lane TP contains 0.25 pl iOE33 translation product. Lanes 1-5 contain 6.25 pg chlorophyll of thylakoid membranes from the binding assay samples.














LL9 e29
A


DMSO BS3 MBS APDP






X-Linking Immunoprecipitation


Figure 2-9. Complex formation with different precursors. A. Crosslinking studies with a variety of precursors and crosslinking agents. The anticpSecA immunoprecipitation products of the cross-linking complex from the thylakoid membrane are shown. ImM cross-linkers BS3 (Bis [sulfosuccinimidyl] suberate),or MBS (m-Maleimidobenzoyl-Nhydroxysuccinimide ester), or APDP (N-[4-(p-Azidosalicylamido) butyl]-3'-[2'pyridyldithio] propionamide) were used for the samples in panels as marked. DMSO is the solvent used for preparing the cross-linkers MBS, APDP. tPC is a truncated form of the pPC precursor and is transported by the cpSec pathway (Henry et al. 1997); iOE23 is transported by the Delta pH pathway. LHCP is integrated into the thylakoid membrane by the chloroplast SRP pathway. B. Crosslinking with precursors that bind tightly to thylakoids. tOE17 is a truncated form of iOE17 and is transported exclusively by the Delta pH pathway; DT33 contains a dual targeting signal peptide and is transported by the cpSec and the Delta pH pathway; DT17 also contains the dual targeting signal peptide, but is only transported by the Delta pH pathway. Binding assays were as described above and contained apyrase (2 units/75 Il assay). DSS was added to the crosslinking reaction at ImM. The cross-linking complex formed on the thylakoid membrane and the anticpSecA immunoprecipitation product of the complex are shown in the left and right panels, respectively.








passenger proteins, although readily transported by the Delta pH pathway, are not translocated by the cpSec pathway (Henry et al. 1997). In addition, these later proteins bind tightly to the membrane. As shown in panel B, total membrane crosslinking products were obtained for tOE17, DTI7, DT-23 (not shown), and DT33. However, CpSecA-containing crosslinking products were only obtained for iOE33 and DT-33. Thus, only those precursors that can be transported by the cpSec pathway, produced cpSecA-containing complexes.



Discussion

Transport of proteins on the thylakoidal cpSec pathway requires stromal protein, specifically cpSecA, ATP, and is stimulated by a transmembrane ApH. Protease treatment of thylakoids eliminates their ability to translocate proteins, indicating that thylakoid membrane bound proteins are also involved. With regards to precursor requirements, a Sec-type signal peptide is essential and a compatible mature protein domain is important for efficient transport. Here we have confirmed previous results of Haward et al. (1997), that cpSec transport can occur in two stages: the first is precursor binding to the membrane and formation of a complex with cpSecA. The second step is translocation across the membrane. We have characterized the binding and translocation steps in some detail, determined at which of these steps the above transport requirements are employed, and examined the stage at which precursor sequences are involved. Our analyses confirm Haward et al.'s (1997) observation that iOE33 formed a productive thylakoid-bound intermediate during the transport when apyrase is present to eliminate ATP from the reaction. Furthermore, our results confirm that the intermediate contains cpSecA. We








show here that cpSecY is also present in the complex, providing the first evidence that cpSecY plays a role in cpSecA-dependent protein transport. cpSec precursor binding to the thylakoid membrane requires cpSecA, and accumulation of the bound precursor is resulted from transport inhibition in the absence of ATP. Subsequent transport of the bound precursor requires ATP, but was not stimulated by additional cpSecA. Precursors capable of being transported by the cpSec pathway were crosslinked into the cpSecA contained complex, whereas precursors transported by the Delta pH pathway and SRP pathways were not.

As noted by Haward et al. (1997), binding does not require ATP, but occurs maximally in its absence. In our experiments, it was necessary to deplete ATP from assay mixture with apyrase to obtain maximum binding to the membrane. We also tried other methods for arresting the transport and promoting precursor binding such as nonhydrolyzable ATP analogues, ionophores, and azide. But so far, apyrase is the only reagent that could promote precursor binding and the complex formation. The apyrase function for arresting precursor transport is presumed to be its ATPase activity that depletes ATP in the reaction. Boiled apyrase lost its function for stimulating precursor binding and the complex formation (data not shown). The effect seen by removing ATP was a specific one because simply inhibiting the transport by dissipating the pH gradient or adding sodium azide were ineffective in promoting binding and had little effect on binding even when ATP was depleted from the assays. Binding of Sec precursor proOmpA to the E. coli membrane also occurred in the absence of ATP (Cunningham et al. 1989). Bound iOE33 was transported from the bound state into the lumen upon resupply of ATP. It is surprising that a ApH did not stimulate the chase reaction. This could








explain, in part, the relatively low percentage of bound precursor that is subsequently transported. In a typical transport assay, the ApH is responsible for -70% of the transport of iOE33. In E. coli, the last step of proOmpA transport could be achieved by either SecA related ATP hydrolysis or PMF (proton motive force) alone in the absence of ATP and SecA (Schiebel et al. 1991). For unknown reason, it seems that the bound iOE33 could only be chased into thylakoid lumen with cpSecA related ATP hydrolysis. A ApH actually reduced the chase because in the presence of ionophores, that eliminated the delta pH, the chase was stimulated. A recent report indicates that PMF facilitates deinsertion of SecA from E. coli membrane (Nishiyama et al. 1999). A ApH might undergo similar function and result in less chase because release of cpSecA from thylakoid membrane.

Chloroplast SecA (cpSecA) facilitates OE33 transport into the thylakoids (Yuan et al. 1994b, Nakai et al. 1994). The fact that most cpSecA is localized in stroma implies that cpSecA functions at early stage of precursor transport which starts in the stroma and ends in the thylakoid lumen. Indeed, cpSecA stimulated precursor binding (Figure 2-2A). The cpSecA was involved in the precursor binding was also suggested by azide treatment which slightly reduced precursor binding and subsequent chase. Azide is a characteristic inhibitor of the ATPase activity of SecA (Oliver et al. 1990). Of interesting is that chase of bound precursor into the thylakoid lumen was not stimulated by soluble pSecA (Figure 2-4). In E. coli, SecA promotes precursor translocation by undergoing ATPdriven cycles of membrane insertion and deinsertion. Each cycle of SecA insertion and deinsertion drives 20-30 aminoacyl residues of the precursor across the membrane (Economou and Wickner 1994). So, complete translocation of a precursor requires








multiple cycles of SecA insertion and deinsertion. It was suggested that after the deinsertion the membrane bound SecA might exchange with soluble SecA or reenter the insertion cycle. From our work, because soluble cpSecA could not stimulate the chase, it seems that cpSecA engaged in the precursor binding/transport maintains contact with the membrane during the precursor chase. This is consistent with the results of Chen et al. (1996) for E. coli SecA. The soluble cpSecA's inability to stimulate the chase also implies that, during transport, cpSecA binds to the thylakoid membrane before precursor does as demonstrated in E. coli system by Cunningham et al. (1989).

The bound precursor could be crosslinked into a high molecular weight complex. Since the complex formation required cpSecA and the complex contained cpSecA and cpSecY, it is called cpSec complex. The detection of such a cpSec complex with bound precursor was an indication that the precursor was engaged in the transport machinery. The idea was further supported by the fact that the precursor binding on the thylakoid membrane was mediated by the protein-protein interactions (Figure 2-5). The results are consistent with those regarding E. coli SecA, that binding between precursor and SecA is very weak in solution, that a strong complex is formed between precursor and SecA on the membrane, and that this complex also contains cpSecY. My results differed from those of Haward et aL (1997) in two respects. First, they obtained a 1:1 crosslinking product between cpSecA and precursor. My crosslinking product was a large migrating product that contains both cpSecA and cpSecY. For the most of my work, crosslinker DSS was used at 1 mM while they used DSS at 0.1 mM or less. A lower concentration of cross-linker might reserve individual cross-linking products which may merge to a larger product with other components when higher concentration cross-linker was used. The








reason for higher concentration cross-linker usage in our experiments was that I could have higher yield of cross-linking product interested. Haward et al. had also obtained a large cross-linking product, but may not show it in their immunoprecipitation. Second, they did not find cpSecY in their complex. Characteristics of their antibodies were not reported. It does not know if their antibodies recognize cpSecY during immunoblotting or immunoprecipitation. In this study, all of antibodies raised against cpSecY immunoprecipitate cpSecY in vitro translation product. The pea C-terminal antibody also recognizes cpSecY during immunoblotting (Mori and Cline, unpublished).

Presumably, cpSecY functions in thylakoid transport in a manner similar to SecY in bacteria, forming part of the membrane translocon. The present results showing that cpSecY was a component of the crosslinked cpSec complex and provides evidence that cpSecY functions with cpSecA in an analogous manner as the bacterial homologues. Recent results from Mori and Cline (unpublished data) demonstrate that antibodies against cpSecY synthetic peptide inhibit protein transport on the cpSec pathway. The cpSecY antibodies inhibited iOE33 binding, indicating that cpSecY was involved in the binding step. iOE33 could either bind to a component associated with cpSecY or bind directly with cpSecY. The antibodies could reduce the binding by disturbing the indirect or direct association between iOE33 and cpSecY.

To know the cpSec complex size is important because it will give information about the complex composition. From the results shown above, the cpSec complex contains at least cpSecA, cpSecY and precursor. CpSecA may work as a dimer. So, the complex could be at least about 300 kD. When the complex was analyzed with SDS-PAGE, it migrated on the gel in a somehow abnormal way. It remains on the top of the separating








gel when a acrylamide concentration higher than 3.5% was used, and it appeared as a smeared band on a 1-16% gradient gel. Under the same conditions, molecular weight markers (67-669 kD) ran as discrete bands. I do not quite understand the complexes' behavior on the gels. The cross-linked complex may represent a mixture of multiple products. In E. coli, the Sec translocase contains more than 9 sub-units (Duong and Wickner 1997). Part of our cross-linking products could contain all the components in the thylakoid transport machine and other parts could contain only several components of the machine.

CpSec complex formation was precursor specific (Figure 2-9). Our analysis shows that precursors destined for the Delta pH translocation system do not enter into the cpSec complex. Even when Delta precursors bind strongly to the membrane and can be crosslinked to thylakoid membrane proteins, they fail to be crosslinked to cpSecA. Of special interest are chimeric precursors that consist of a dual targeting signal peptide linked to a Delta pH passenger protein. These proteins also fail to be crosslinked to cpSecA. Previous studies have shown that the DT signal peptide is capable of directing efficient cpSec transport when coupled to a passenger protein that is normally transported on the thylakoidal Sec pathway, but not when linked to Delta pH passenger proteins (Henry et al. 1997). This suggests that these proteins may not be recognized by cpSecA, possibly because they cannot adopt a compatible loop for the cpSecA system. It has been shown in E. coli that the first 20-30 residues of the mature protein sequence play an important role in transport. When unpaired basic residues are introduced into this region, it poisons translocation. Delta pH precursors do contain significant numbers of basic residues in their N-termini. Support for this notion comes from recent experiments that








show that OE23 can be efficiently translocated across the Sec pathway when fused to the pPC transit peptide plus 25 residues of mature domain (Cline, unpublished).

A primary interest in my study was to determine the step or reaction that commits a precursor to the cpSec pathway. SecA or SecY in E. coli Sec system are considered to be receptors. Since the first detectable interaction between precursor and components of the cpSec pathway machinery was on the membrane, I assume that recognition occurs on the membrane. The interaction on the membrane could involve primarily cpSecA given the fact that about 20% epSecA in chloroplasts was on the thylakoid membrane (Yuan et al 1994). Two lines of evidence from my experiments support the notion that cpSecA on the membrane could function as a receptor. Firstly, cpSecA stimulates precursor binding to the membrane and the bound precursor is associated with cpSecA. Secondly, since soluble cpSecA does not stimulate precursor chase cpSecA must bind to the thylakoid membrane before or at the same time as precursor does, which is consistent with a receptor role. cpSecY is also involved in precursor binding and is part of the cpSec complex, which supports its role as a receptor. DT33, DT23 and DT17 all could be crosslinked to cpSecY (data not shown), but only could DT33 be cross-linked to cpSecA. It is possible that cpSec-compatible precursors can directly bind to cpSecY as ER precursors can directly bind to Sec61, but strong binding and subsequent transport requires binding to cpSecA. Because DT-23 and DT-17 appear to bind cpSecY, but not to cpSecA, then it could be that the block in cpSec transport of these precursors is their inability to bind cpSecA.





53

In summary, my results are consistent with the model that thylakoidal Sec substrates are committed to the Sec pathway on the membrane and that the initial region of the mature protein as well as the signal peptide plays a critical role in that reaction.













CHAPTER 3
PROTEIN TARGETING AND TRANSLOCATION ON THE DELTA pH PATHWAY Abstract

The Delta pH pathway is one of two systems for protein transport into the thylakoid lumen. This pathway requires only the trans-thylakoid pH gradient to power translocation. A newly constructed precursor protein (tOE17), that uses the Delta pH pathway, binds to the membrane in substantial amount in the absence of the ApH. Binding is productive because bound precursor is transported into the lumen when the ApH is restored. Several observations suggest that binding is due to protein-protein interactions. Protease pretreatment of thylakoids reduced their ability to bind precursor; precursor binding was sensitive to salt concentration; and productive binding was competed by saturating amounts of a Delta pH pathway precursor protein. These results suggest that precursor tOE17 binds to components of the Delta pathway translocation machinery. Transport from the bound state, which we refer to as chase, was very efficient

- up to 90% of bound precursor could be chased into the thylakoid lumen. Consistent with the transport requirements, the ApH was the only energy source necessary for the chase reaction. The identification and characterization of a productively bound intermediate in this study provide biochemical tools for detecting the nature and function of components of the Delta pH pathway. Antibodies raised to E. coli-expressed Hcfl06, when pre-bound to thylakoids, specifically inhibited transport on the Delta-pH pathway and reduced the








level of productive binding. The results suggest that Hcfl06 functions in early steps of the transport process, possibly as a receptor.



Introduction

The Delta pH pathway was first recognized as a distinct transport pathway by the discovery that different subgroups of precursors had different energy and soluble protein requirements for translocation. Specially, transport of precursors OE17 and OE23 required neither soluble protein factors nor NTPs. Their transport depended entirely on the trans-thylakoid ApH (Cline et al. 1992). A requirement for specific translocation machinery was further demonstrated by precursor competition studies (Cline et al. 1993). In a competition assay with thylakoids, saturating amounts of iOE23 selectively competed with iOE17 on the same pathway but not with iOE33 on the other pathway.

Although the Delta pH pathway was identified years ago, knowledge about its mechanism is quite limited. One reason is that intermediate steps of process had not been identified. Identification of intermediates in other systems directly led to identification of components. The import of protein across the chloroplast envelope is one excellent example (reviewed by Cline and Henry 1996, Schnell 1998). In the presence of low concentration of NTPs (50-100 iM), protein import into chloroplast is arrested, resulting in bound precursor on the envelope membrane. When the ATP concentration is raised to approximately 1 mM, the bound precursor is translocated into chloroplast. Bound intermediates were then used to identify import machinery components (Schnell and Blobel 1993, Schnell et aL 1994, Kessler et al. 1994, Ma et al. 1996). The ability to arrest productively bound precursor on the thylakoid cpSec pathway (see chapter 1 in this








dissertation and Haward et al. 1997) led to identification of components and precursor elements in the targeting process. Biochemical approaches, such as cross-linking and native gel techniques to identify machinery components have not been successful for the Delta pathway system because intermediates have not been produced.

In this study, certain truncated Delta pH pathway precursors were observed to bind tightly to the thylakoid membrane in the absence of ApH, and the bound precursors were transported into the thylakoids after the ApH was restored. The characteristics of precursor binding and chase process will be described.



Materials and Methods

Preparation of Radiolabeled tOE17 by in vitro Translation The coding sequence of tOE17 was amplified with the forward primer 5'AATTATGGCGGGCCGCCGCGC-3' and reverse primer 5'GTT1TCCCAGTCACGAC-3' (Nebl212.Pri) using a pGEM7zf plasmid containing maize pOE17 as a template. The PCR product was restricted with Xbal and ligated into XbaI and Smal restricted pGEM 4Z. Capped RNA for tOE17 was produced as described by Cline (1988). RNA was translated in a wheat germ system (Cline et al. 1989) in the presence of [3H]-leucine.

Preparation of Other Precursors

In vitro transcription plasmids for iOE33, iOE23 and LHCP were described as Cline et al. (1993). Preparation of tOE23, iOE17, iPSAN, DTPC, DT17, DT23, DT33 were described as Henry et al. (1997). Preparation of HyaPC was described as Mori et al. (1998).








Over-expression of tOE17, DT23 and pOE33 in E coli

The coding sequence of tOE17 was amplified with the forward primer 5'CGTTGCGGGATCCGGCCGCCG-CGCCGTGATCG-3' and reverse primer 5'CCATTATTAGAATTCGCGTCTAGCCTAGCTTGGCG -3' using the pOEl7 plasmid as a

template. The PCR product was restricted with BamHI and EcoRl and ligated into appropriately restricted pGEX-2T plasmid. tOE17 was expressed as a glutathione S transferase (GST) fusion protein in E. coli. Expression, purification and digestion of the fusion protein with thrombin followed the BRL company instructions. The final tOE17 product after thrombin cleavage was two amino acids different from the in vitro translation product. Instead of the amino acids MA, E. coli-expressed tOE17 has the amino acids GS at its amino terminus. E. coli-expressed DT23 and pOE33 were produced as described respectively by Henry et al. (1997) and Cline et al. (1993). Preparation of Chloroplasts, Lysates, Thylakoids and Stroma

Pea chloroplasts were isolated from 9-10-day-old seedling; lysates, thylakoids and stromal extract were prepared from intact chloroplasts as described by Cline (1991). Thylakoid Protein Transport Assay

Transport assays were conducted in 1.5 ml microcentrifuge tubes. In vitro translation product (0.5 to 5 pl) or 0 to 2 ipM E. coli- expressed precursor was incubated with 12.5 pg chlorophyll of thylakoids or chloroplast lysate in a total volume of 37.5 p l of import buffer (50 mM Hepes buffer pH 8, 0.33 M sorbitol). Transport reaction was conducted in 70 jiE m-2s%' white light for 15 minutes at 250C. After incubation, thylakoids were recovered by centrifugation and washed with import buffer. Final membrane samples








were either analyzed directly on SDS-PAGE or treated with 10 jIg thermolysin in 100 Il of import buffer (Cline, 1986) and then analyzed by SDS-PAGE and flurography. Precursor Binding and Chase Assays

Ten Il [3 H] labeled in vitro translated precursor was incubated with 25 jIg chlorophyll of thylakoids or chloroplast lysate in the presence of 3 M CCCP or 2 units of apyrase for 15 minute at 0OC in a total volume of 75 il of import buffer. The thylakoid membranes were then pelleted at 2500x g, washed with import buffer two times, and divided into two equal portions. One portion was directly analyzed to access the amount of precursor bound to the thylakoid membrane. The second portion was incubated under light for 15 minutes at 250C in a total of 37.5 pl of chase buffer mixture, which contained

-200 ig protein of stromal extract and I mM DTT. In some assays, thylakoids recovered from binding or chase assays were treated with 10 jg of thermolysin in 100 il of import buffer (Cline 1986).

Preparation of Thermolysin- or Proteinase K- Treated Thylakoids for use in Binding Assays

Thermolysin-treated thylakoids were prepared as following: 0.5 jg chlorophyll/ld of thylakoids were incubated with 0-160 jg thermolysin/ml from a 2 mg/ml stock containing 10 mM CaCI2. The incubation was in import buffer on ice and lasted for about 60 minutes. The thylakoid membranes were then recovered by centrifugation and washed twice with import buffer containing 10 mM EDTA and once with stromal extract (containing -2.5 jtg protein/!Il) in import buffer. The washed thylakoids were then resuspended in import buffer at 1 mg chlorophyll/ml. The proteinase K treatedthylakoids were prepared as following: 0.5 jg chlorophyll/Il of thylakoids were








incubated with 0-80 jig protease K /ml from a 1mg/ml stock. The incubation was in import buffer on ice and lasted for about 60 minutes. PMSF (Phenylmethylsulfonyl Fluoride) was added to 5 mM and the incubation was continued for an additional 10 minutes on ice. Thylakoid membranes were then recovered by centrifugation and washed twice with import buffer containing 2 mM PMSF and once with stromal extract (-2.5 lg/ll) in import buffer. The washed thylakoids were resuspended in import buffer at

1 mg chlorophyll/ml.

Analysis of Samples

Samples recovered from the assays were subjected to SDS-PAGE and fluorography (Cline 1986). Quantification of the amount of transport, binding or chase was accomplished by scintillation counting of radiolabeled proteins extracted from excised gel bands (Cline 1986).



Results

Dissipating the ApH across Thylakoids Arrested tOE17 Transport and Resulted in Accumulation of a Transport Intermediate on the Thylakoid Membrane

Protein transport into the thylakoid lumen is presumed to start with a targeting event in which the precursor interacts with a transport machinery component. For the Delta pathway, precursor targeting undoubtedly occurs on the thylakoid membrane because soluble components are not required for transport. However, before this study, stable precursor binding to the thylakoids had not been observed. I surveyed a number of precursors, both authentic and recombinant, and found several ones that bound to the thylakoid membrane in the absence of ApH (see below).








tOE17 is an N-terminal truncated form of iOE17 that I constructed as part of another study on targeting determinants (Henry et al. 1997). All thylakoid precursor proteins contain lumen-targeting domains (LTD) which can be divided into four sub-domains: an acidic domain (A), a charged domain (N), a hydrophobic domain (IH), and a cleavage domain (C) for proteolytic processing. tOE17 contains only the N, H and C domains. While the H, C domains and the followed mature part are exactly the same as iOE17, the N domain in the far end of tOE17 contains only MAGRR amino acid residues. According to Henry et al. (1994, 1997), N, H and C domains in the LTD are sufficient for specific precursor targeting and transport. Unexpectedly, truncation of iOE17 produced our most efficient Delta pH pathway transport substrate. Figure 3-1 shows transport assays conducted with tOE17 under varying assay conditions. The assay was started by incubating radiolabeled tOE17 in vitro translation product with the purified thylakoids. Upon transport into the thylakoid lumen, the LTD is removed from the precursor, resulting in the smaller mature OE17. Figure 3-1 demonstrates that tOE17 transport was inhibited when the ApH across thylakoid membrane was either dissipated by a combination of ionophores, nigericin and valinomycin, or prevented from forming by incubation in the dark with apyrase to deplete ATP. This figure also shows that tOE17 exhibits transport conditions identical to authentic Delta pH pathway precursors, i.e. it occurs in the absence of ATP or stroma, and it was absolutely dependent on the pH gradient.

Although tOE17 transport was inhibited by depleting the ApH, a substantial quantity of precursors remained bound to the thylakoid membrane. Bound precursor was degraded by protease treatment of thylakoids, demonstrating that it was exposed on the exterior of















ApH - + -+ + +
light - + + + + +
ATP - + +- + +
ionophores - - + - -
stroma + + + + - +
apyrase + - -+ -
azide - - - - - +
P1 2 34 5 6
p O


Protease A 0
posttreatment



Figure 3-1. tOE17 transport was inhibited by dissipating the ApH, resulting in accumulation of bound precursor on the thylakoid membrane. Ten g1 [3H]-leucine labeled tOE17 precursor was incubated with 25 gg chlorophyll of chloroplast lysate or isolated thylakoids (lane 5) in a total of 75 jil of import buffer. The assays were conducted under conditions designed to test the effects, on binding and transport, of removing ATP or stromal extract, of dissipating the ApH, or of inhibiting cpSecA with azide. Stromal extract was about 400 jig of protein per 75 pl assay, Mg-ATP was added to 5 mM final concentration, the ionophores nigericin and valinomycin were added to 0.5 jM and 1 pM, respectively, from ethanolic stocks. Apyrase treatment was 2 units /75 pl assay. Azide was added to 10 mM final concentration. After incubation at 25cC for 15 minutes the thylakoid membranes were recovered by centrifugation, washed with import buffer two times, and divided into two equal portions in new microfuge tubes. One portion was directly analyzed with SDS-PAGE and fluorography (top panel). The second portion was first treated with protease and then analyzed by SDS-PAGE and flurography (bottom panel). Each lane contained 12.5 jg chlorophyll of thylakoid membranes. Lane P represents 0.25 gl (5%) precursor added to the assay reaction. The positions of the precursor tOE 17 (p) and the mature form (m) are marked.








the thylakoids. The mature OE17 was resistant to protease treatment confirming its presence in the thylakoid lumen.

Bound Precursor is a Productive Intermediate because It can be Chased into the Lumen when the ApH is Restored

If the tOE17 binding is physiologically significant, then bound precursor should proceed into the thylakoid lumen once the ApH is restored. I refer to this transport from the bound state as "chase". Binding and chase assays were conducted as shown in Figure 3-2. Radiolabeled precursor was incubated with thylakoids in the dark in the presence of the ionophore CCCP (Carbonylcyanide 3-chlorophenylhydrazone) or with apyrase at 0oC for 15 minutes, during which time tOE17 precursor bound to the thylakoid membrane. Then unbound precursor and ionophore or apyrase were washed away with import buffer and the thylakoids with bound precursor were resuspended in buffer containing stroma and DTT (dithiothreitol) and transferred to 250C and white light. Under light the bound precursor was chased into the thylakoids. The time required for total chase was -8 minutes. About 5.6-9.4% of the added precursor to the assay was bound to the thylakoid membrane and -80% of the bound precursor chased into the lumen. The results indicated that the bound precursor is a productive intermediate on the Delta pH pathway.

Other precursors that can be transported on the ApH pathway, most of them made in Cline's laboratory, were also tested in the binding and chase assays. As shown in Figure 3-2 (B), the ability to bind tightly to the thylakoid membrane varied among these precursors. tOE17, DTI7 and DT23 bound strongly to the thylakoid membrane and a substantial percentage of bound precursor was chased into thylakoids. iOE17 exhibited moderate binding and some bound was chased into thylakoids. HyaPc or iPSAN








Bind Chase
A P 1 2 3 4 5 6
Binding with -4apyrase

Binding with -
CCCP
cccP
Protese - - + - - +
post-treatment

Bind Chase Bind Chase Bind Chase B P 234 P1 23 4 P1 2 3 4
PAS g iOE7 dtOE17

tOE23 iOE23 dtOE23

HyaPC dtPC dtOE334*

Protease + + + . + - + - +
post-treatment


Figure 3-2. Precursor binding-chase experiments. A. Seventy five tg chlorophyll of thylakoids were incubated with 30 gl [3H]- labeled tOE17 in vitro translation product in darkness in the presence of 3 pM CCCP or 6 units of apyrase in a total 225 tl of import buffer. The incubation was for about 15 min on ice. The thylakoid membranes were washed with import buffer, divided into 6 portions, and transferred to new tubes. One portion was analyzed directly on SDS-PAGE (lane 1). The second and third portions were treated with thermolysin (lane 3) or mock-buffer (lane 2) and then analyzed on SDS-PAGE. The remaining portions were incubated for chase into the lumen. The chase started with the recovered thylakoids from the binding incubation. The thylakoids were resuspended in 37.5 pl IB containing about 180 pg protein of stromal extract and ImM DTT (dithiothreitol). The resuspended thylakoids were transferred to 70 pE.m2s' white light and incubated for 15 minutes at 25�C. Chase samples were then analyzed directly (lane 4), or treated with thermolysin (lane 6) or mock-buffer (lane 5) and then analyzed on SDS-PAGE. Each lane was loaded with 12.5 pg chlorophyll of thylakoids from each assay. Lane p represents 0.25 pl of the precursor added to the assay reaction. B. The assays were conducted as described above with a variety of precursors that can be transported on the Delta pH pathway. Binding assays were in the presence of 3 pM CCCP. Lane 1, bound precursor on the washed thylakoids; lane 2, thermolysin treated binding samples; lane 3, chase samples; lane 4. thermolysin treated chase samples. Lane p represents 0.25 pl of the precursor added to the assay reaction.








exhibited substantial binding but the bound HyaPC and iPSAN were not subsequently chased into the lumen. There was hardly any binding for tOE23, iOE23, DT33 and DTPC. The reason that some precursors can bind better than others is not clear. The binding ability appears to be related to both precursor LTD and the mature protein sequence. More information about these precursors can be found in papers published by Henry et al. (1997) and Mori et al. (1998).

I have examined several ways of dissipating the ApH in the dark as a means of accumulating bound precursor. These included apyrase treatment to scavenge all traces of ATP, which could be hydrolyzed by proton-pumping CFI/CFo to generate ApH. These also included CCCP treatment. Three pM CCCP was the optimal concentration for arresting the transport and allowing chase. Lower CCCP concentration did not completely arrest transport. Higher CCCP concentration resulted in less binding and did not permit chase. CCCP has the advantage of being water soluble, such that it can be washed from the thylakoid membranes. It is possible that high levels of CCCP cannot be adequately removed by washing or, alternatively, that the high level of CCCP compromises membrane integrity. The ionophore combination of nigericin and valinomicin resulted in high levels of precursor binding, but the bound precursor was not chased. This is consistent with the fact that nigericin and valinomycin are lipid soluble and probably persist though washing steps. Initially, the binding assay was conducted at room temperature. Subsequent tests indicated that binding under icy temperature was comparable to that at room temperature, resulted in a higher percentage of chase. I found it necessary to eliminate the ApH, even when binding assays were conducted at 0OC. Because transport of some precursor on the Delta pH pathway is so efficient that it occurs








at icy temperature (Figure 3-3). Precursor binding and subsequent chase on the Delta pH pathway occurred in the presence or absence of stromal extract, so stroma was not required for productive binding (data not shown).



Chase Requirements for the Productively Bound tOE17

The ApH across the thylakoid membrane is the only energy requirement for protein transport on the ApH pathway. Although I assumed that transport of bound tOE17 occurred on the Delta pH pathway, it was important to examine the chase requirements. A chase requirement assay is reported in Figure 3-4. The chase was inhibited by either nigericin and valinomycin or dark plus apyrase treatment. Apyrase treatment in light did not inhibit the chase. These results indicate that ApH is required for the chase. Buffer containing Mg++ resulted in higher percentage of chase than buffer alone. As expected, ATP did not stimulate the chase. Stromal extract was not required for the chase, but had some stimulation effect. I also examined the chase requirements for DT-23. This precursor has a dual targeting signal peptide, but is only transported on the Delta pH pathway. Bound DT-23 was also transported exclusively by a Delta pH pathway reaction.



tOE17 Binding is Due to Protein-Protein Interaction Suggesting that the Bound Precursor be Associating with Some Machinery Component on the Transport Pathway

The binding-chase experiment demonstrated that the binding is productive. However, I did not know whether the binding is specific or not. Specific binding should exhibit several characters: (1). the bound precursor should be specifically associated with a specific component, probably a protein component of the transport machinery; (2). the














OoC 250C 0CC 25cC 0OC 250C
p1 2 3 4 p 12 34 p 1 234 tOE 17 iOE17 1DTI7

tOE23 iOE23 DT23
LHCP iOE33 DT33 HyaPC # - DTPC
Protease
post-trtment - +- +_ + - + - +




Figure 3-3. Substantial transport could be achieved on the Delta pH pathway at icy temperature for some precursors. Nine ll in vitro precursor was mixed with 25 pg chlorophyll of choroplast lysate, 200 gg protein of stromal extract and 5 mM ATP in a total 75 l of import buffer. Incubation was conducted either in icy water (00C) or 25�C in white light. After incubation for 20 minutes, samples were transferred to an ice bath and 0.5 gM/1 jM ionophores nigericin/ valinomycin were added. Thylakoid membranes were recovered by centrifugation, washed with import buffer two times, and divided into two equal portions in new microfuge tubes. One portion was directly analyzed with SDSPAGE and fluorography (Lanes 1 and 3). The second portion was first treated with protease and then analyzed by SDS-PAGE and flurography (Lanes 2 and 4). Each lane contained 12.5 jg chlorophyll of thylakoid membranes present in each assay. Lane P represents 0.25 pl precursor added to the assay reaction.












Chase Conditions



0 go
0 6 < Chase Conditions

P 1 2 3 4 5 6 7 8 9

I <
Pl12 3 4 5 6 7 8




A B

Figure 3-4. Chase requirements for the productively bound tOE17 and DT23. A. 225 pg chlorophyll of thylakoids were incubated with 90 al '[H]-labeled tOE17 in vitro translation product in darkness in the presence of 3 gM CCCP for about 15 min on ice. The thylakoids were recovered by centrifugation and washed one time with import buffer. The washed thylakoids were resuspended in import buffer, divided into 9 equal portions and transferred to new tubes. One portion was used to assess the amount of bound precursor (lane 1)--- half of the thylakoid membranes was directly resuspended in SDS sample buffer; the other half was treated with protease thermolysin before resuspending in SDS sample buffer. The remainimg portions received further chase treatments under conditions designed to test the effects of ATP, ApH, stromal extract
(SE) and Mg++. Stromal extract was present at about 375 gg protein. Mg++ or Mg-ATP was added to 5 mM final concentration. Nigericin and valinomycin (N/V) were added to 0.5 and 1 M, respectively, from an ethanolic stock. Apyrase was added at 2 unit/75 gl assay. The chase assay was conducted under 70 tE.m'2s-1 white light (darkness for lane 8) for 15 min. at 250C. Finally, half samples were treated with thermolysin (low panel); another half samples were mock-treated with import buffer (upper panel). Lane p contained an aliquot of the precursor added to the binding incubation. B. Experiments were conducted similarly as above but precursor DT23 was used. Samples were treated with thermolysin after the chase.








binding should be satuable; (3). The binding should be competed by precursors known to use the same pathway.

I tested if the binding involved a protein-protein interaction (Figure 3-5). tOE17 was incubated with protease-treated thylakoids and assayed for binding or transport. Bound precursor was subsequently analized for chase. Before incubation with precursor, the protease-treated thylakoids were thoroughly washed with buffer containing protease inhibitors in order to eliminate any residual protease from the preparation. Precursor binding to the protease-treated thylakoids was substantially reduced compared to control (buffer washed thylakoids). Proteinase K pretreatment had less of an effect on precursor binding than thermolysin treatment. The subsequent chase of any bound precursor on both protease treated thylakoids was totally inhibited indicating that both protease treatments damaged the transport system. This was also demonstrated by assaying the treated membrane for transport with fresh precursor. It is of interest that thermolysin effects on binding and transport are highly correlated. The nature of the precursor binding was also assessed by urea and sodium carbonate washes. Proteins that are embedded in the lipid phase of a membrane are not extracted by these wash treatments, whereas extrinsic or peripheral proteins that are associated with membranes via protein-protein interaction are removed (Fujiki et al. 1982). All bound precursors on the thylakoids were extracted by the 0.1M sodium carbonate and most of the bound precursors were extracted with 6M urea washes (data not shown). These results further suggest that the precursors bind directly to membrane proteins under experimental conditions.









Protease pretreatment of the thylakoids Thermolysin Proteinase K
"P 0 %P (z''-, p0%mgA1) P 1 2 3 4 5 6 7 8 9 10 A. Binding -g .m


B. Chase
me a

C. Transport q1


D. Binding
mixture " *

E. Transport qi ) W g O mixture iet m d *



14000 1 14000
12000 12000 - Binding
- Chase
10000 - Binding 10000 Transport
sooo- Chase soo
8000- ahO 80001
6000- Transport 6
E 60004000 4000
20002000
------ -20000
0 20 40 80 160 0' 1,
Thermolysin (mg/I) 0 10 20 40 80 Proteinase K(mg/iI)



Figure 3-5. tOE17 binding on the thylakoid membrane is mediated by protein-protein interactions. 3H-labeled tOE17 in vitro translation product was incubated with thermolysin- (0-160 tg/ml) or proteinase K- (0-80 gtg/ml) treated thylakoids in the presence of 3 tM CCCP ionophore. Binding, chase and transport assays were conducted essentially as previously described. Panels A, B and C are the buffer-washed thylakoid membranes samples after binding, chase and transport assays. To confirm that no residual protease remained in the reactions, aliquots were removed from binding and transport incubation mixtures and analyzed directly on SDS-PAGE . As shown in Panels D and E, free precursors in the solution were not degraded, indicating that the pre-protease treated thylakoids did not contain any residual protease.








The tOE17 Binding is Salt Sensitive

The transit peptides in Delta pH pathway precursor contain a twin-arginine motif that is crucial for the transport pathway selection (Chaddock et al. 1995). One possibility is that the positive charges in the motif may be involved in the interaction between precursor and a putative receptor. If so, then binding should be sensitive to salt concentration. The binding assays were conducted in the presence of increasing concentrations of KC1. As shown in the Figure 3-6, precursor binding in the presence of higher concentration of KCl was decreased as was the subsequent chase. Transport, however, was less influenced by KC1. Transport actually increased slightly from 0-150 mM KCl and decreased at further higher concentration of KC1.



Productive Binding can be Competed by Delta pH Precursors

If the precursor binding on the thylakoid membrane is an interaction between precursor and a receptor-like component, binding should be saturable and competible. To test this, binding of radiolabeled in vitro translated tOE17 was conducted in the presence of increasing concentrations of unlabeled E. coli-produced precursor proteins (Figure 37). The presence of increasing concentration of unlabeled tOE17 precursor resulted in decreased radiolabeled precursor binding, although there remained residual binding that actually increased at high concentration of unlabeled tOE17. Importantly, the amount of productively bound precursor decreased dramatically. Productively-bound precursor was determined as that which chased into the lumen upon favorable conditions. From the chase experiment, it is significant that competition for productive binding follows a similar inhibition curves as competition for transport of freshly added precursor. Thus,








KCI (mM)
P
A. Binding


B. Chase


C. Transport



Figure 3-6. tOE17 binding in the presence of increasing concentrations of KCl (0500mM). Panel A and panel B are binding-chase assays. Eight tubes, each of which contained 10 pl 3H-labeled tOE17 in vitro translation product and 9 pl import buffer, were preincubated with 1 unit (pl) apyrase for 10 minutes on ice and then mixed with 37.5 pl 0, 100, 200, 300, 400, 600, 800, or 1000 mM KCl in import buffer. Twenty five pg chlorophyll of thylakids in 17.5pl import buffer, which was preincubated with 1 unit apyrase, was added to each tube. The final incubation volume of each tube was 75 pl and the KCI concentration was 0-500 mM. After 15 minutes incubation on ice the thylakoid membranes in each tube were washed respectively with 0, 50, 100, 150, 200, 300, 400, or 500 mM KCl in import buffer and then washed with import buffer. The washed thylakoid membranes were divided to 2 equal portions and transferred to new tubes. One portion was analyzed directly on SDS-PAGE for binding (panel A). The second portion was incubated for chase. The thylakoids in final chase samples were treated with thermolysin and then analyzed on SDS-PAGE (panel B). Panel C shows transport f freshly added precursor. Chloroplast lysate (12.5 pg chlorophyll) was mixed with 1.5 pl tOE17 translation product in the presence of 0-500 mM KCl in a total 37.5 pl of import buffer. Lane p represents 0.25 pl of the precursor added to the assay reaction. The other lanes in panel A, B and C were loaded with 6.25 pg chlorophyll of thylakoids from each assay.






72



20000

V 15000 - Binding Unlabeled tOE17( pM) " Chase
10000 Transport P o "
Binding - -*
050000 I I Chase
0 0.25 0.5 1 2 Unlabeled tOE17 Transport -*





Unlabeled dt23 ( pM)
Unlabeled pOE33OM)
P c - ., P1 Binding - finding *
%b a~lig 44PcB

Chase WlO Chase

Transport Transport W0

20000 - Binding 20000 Binding - Chse S15000 - Chase 15000- Transport cTransport C
10000 410000 S5000 5000

0 -0 -
0 0.25 0.5 1 2 0 0.25 0.5 1 2 Unlabeled dt23 Unlabeled pOE33




Figure 3-7. Binding competition. Binding-chase assays started with 10 pl 3H-labeled tOE17 in vitro translation product and 25 jig chlorophyll of thylakoids in the presence of increasing concentrations of unlabeled E coli produced tOE17 or pOE33 or DT23 in a total 75 ll import buffer. Before the adding of radiolabeled precursor, unlabeled precursors were pre-incubated with chloroplast lysate for 3 min on ice. Subsequent chase was conducted with the washed thylakoids after the binding. Results of 3H-OE17 transport in the presence of 0-2 pM E. coli. produced unlabeled tOE17 or pOE33 or DT23 were also shown.








there appears to be two kinds of binding involved. One is productive binding, presumably from an interaction between precursor and transport machinery. Another is nonproductive or non-specific binding. The amount of non-productive binding varied from experiment to experiment. It became a significant percentage of total binding when E. coli.- produced precursors were present in the binding assay. This is demonstrated most dramatically in the tOE17 binding saturation experiment (data not shown). Therefor, the non-productive binding may result from precursor aggregation on the thylakoid membrane. It seems apparent to me from Figure 3-7 that the productive binding is competible and the non-productive binding is not. Similar results of competition for labeled tOE17 binding-chase and transport were also obtained with unlabeled DT23 as competitor. pOE33 is transported on the cpSec pathway. E. coli produced pOE33 did not have any effects on tOE17 binding, chase or transport. The above results demonstrate that elevated concentration of tOE17 saturates a membrane component involving in the precursor binding process.

In Figure 3-7, it is apparent that in vitro translation of tOE17 produces two products. One is tOE17; a second migrates at the approximately location of mature OE17. This lower band, which varies in amount with the translation reaction, also binds to the membrane. It is unlikely that it is mOE17 because there is only one methionine in tOE17 that could serve as initiator and because it is competed by DT-23 as well as tOE17 but not pOE33, indicating that it is being competed by virtue of the targeting sequence rather than the mature sequence. It is not clear how it is produced from the in vitro translation, but may result from early termination, which has previously been observed with wheat germ translation systems (Mori and Cline, unpublished).










Bound Precursor Chase is Competible

We further want to know whether there is any competition for transport from the bound state. The radiolabeled tOE17 was allowed to bind on the thylakoids without competitor. After washing away unbound free precursor the thylakoid membranes with bound precursor were then resuspended in the buffer containing 0 to 2 pM unlabeled tOE17 and a chase reaction was conducted. As shown in Figure 3-8, chase of bound precursor decreased in the presence of unlabeled precursor. The results indicate that in addition to competition for binding the chase reaction can also be competed. We have noticed that a significant amount of bound precursors was chased into the thylakoids even in the presence of high levels of unlabeled competitor. This did not occur in the competition for transport that was conducted as a control with the CCCP treated thylakoids plus freshly added labeled precursor under the same condition as the chase.



tOEl7 Binding is Not Reversible

One possibility for the above chase competition is that during the chase process the bound radiolabeled precursor is released into solution and re-enters the Delta pH pathway as a soluble precursor. In this case, the observed competition for chase would be equivalent to competition for transport of free precursors. To clarify this issue, a different binding competition experiment was conducted to assess the reversibility of binding (Figure 3-9). In the above binding competition tests, chemical amount of E. coli. produced unlabeled precursors were incubated first with thylakoids and the radiolabeled in vitro translation precursors were added afterwards. If radio labeled precursors were










E.coli produced 3H-tOE17 (pIM)
ot *, .E.coli produced 3H-tOE17 (pM)

P 1 2 3 4 5 6 7 8 9 Pl23456789
P 1 2 3 4 5 6 7




Chase Competition Transport Competition


Figure 3-8. Bound precursor chase competition. In the left panel, 112.5 lag chlorophyll of thylakoids were incubated with 45 gl 3H- labeled tOE17 in vitro translation product in dark in the presence of 3 p.M CCCP. After 15 minutes incubation on ice, the thylakoids were recovered by centrifugation and washed with import buffer and divided into 9 equal portions and transferred to new tubes. For checking precursor binding, one portion was directly analyzed with SDS-PAGE (lane 1); second portion was treated with protease thermolysin, and then analyzed with SDS-PAGE (lane 2). The thylakoid membranes in the remaining portions (lane 3-9) were resuspended in buffer containing increasing concentrations of unlabeled E coli produced tOE17 and incubated under chase conditions. Final chase samples were treated with thermolysin. Transport assay controls are presented in the right panel. 12.5 jg chlorophyll of thylakoids, which were pre-treated with 3 p.M CCCP and buffer-washed, were mixed with buffer containing 1 p1l "H- labeled tOE17 in vitro translation product and increasing concentrations of unlabeled E coli produced tOE17 and incubated under chase conditions. Final transport samples were treated with thermolysin. Lane P contains an aliquot of the precursor added to assays. The remaining lanes in both panels were loaded with 6.25 jig chlorophyll of thylakoids from each assay.





















Unlabeled precursor (pM) in binding P o" '" % O

Binding M W - t



Chase m e






9000


7000 6000


- Binding
L40003000

2000 1000

0 -H-- -r- - -- -0 0.25 0.5 1 2 Competitor concentration (micro morlar)



Figure 3-9. Effect of unlabeled precursor on the binding of preloaded radiolabeled tOE17 in vitro translation product. Ten il radiolabeled tOE17 was incubated with 25 pg chlorophyll of thylakoids for 5 min, then 0-2 pM unlabeled E. coli produced precursor (tOE17) was added and the incubation continued for 15 minutes. After washing with import buffer, the membranes were transfer to new tubes. Half of each sample was analyzed directly for the amount of precursor bound to the membrane; remaining half of each sample was incubated for chase in a buffer containing stroma and DTT.








incubated first with the thylakoids, its binding was not competed by the added unlabeled precursor. However, the subsequent chase of the bound radiolabeled precursor was decreased. The results demonstrate that the precursor binding is quite strong and hardly replaced. Also, the results further confirm that there is real competition at chase step, though the competition occurs between the bound precursors.



Hefl06 is involved in the tOE17 binding

Hcfl06 was identified in maize as a component of the Delta pH pathway by both in vivo and in vitro methods (Voelker and Barkan 1995, Settles et al. 1997). Tha4 is a maize homologue of Hcfl06. a cDNA for psTha4, a pea homologue of Tha4, was recently isolated in Cline's laboratory. Antibodies against maize Hcfl06 or psTha4 were prepared (Mori et al., in preparation). The effects of antibodies against maize Hcf 106, pea psTha4 and pea cpSecY, on tOE17 binding-chase and transport to pea thylakoids, were examined. Thylakoids were pre-incubated with either antiserum IgG or preimmune serum IgG. After washing, the thylakoids were assayed for binding-chase and transport of 3Hlabeled tOE 17.

As shown in Figure 3-10, Hcfl06 and psTha4 antibodies inhibited tOE17 transport and chase, indicating Hcfl06 and psTha4 function on the Delta pH pathway. The inhibitions caused by antibody against Hcfl06 could be reversed by antigen Hcfl06 protein. Antibody against cpSecY, which plays an essential role on the cpSec pathway, did not have any inhibition. I have noticed that although both Hcfl06 and psTha4 antibodies specifically inhibited tOE17 transport and chase, only Hcfl06 antibody reduced the binding. Preliminary experiments indicated that the bound tOE17 could form




















S0
P 1 23 45


67 89


P 1 2 3 4 5 Binding .me. .


Chase


-iw
a *


Transport w


Transport I


Antigen


Figure 3-10. tOE17 binding-chase and transport to the thylakoids pre-treated with Hcfl06, psTha4 or cpSecY antibodies. In A panels, 0.33 gg chlorophyll /jl of thylakoids were pre-incubated with 0.5 or 1.0 gg IgG /l of immune or preimmune in 3% BSA-IBM buffer. The incubations were for one hour in an ice bath. Thylakoid membranes were washed once with IB buffer and used for binding-chase and transport assays as describing in methods. In B panels, thylakoids were pre-incubated with 0.8 gg IgG / l of antiHcfl06 or preimmune in 3% BSA-IBM buffer in the presence or absence of 15 JIM HcflO06 antigen. Following steps as A panels. For both A and B panels, Lane P contains 0.25 gl tOE17 in vitro translation product. The remaining lanes contain 6.25 jig chlorophyll of thylakoid membranes from the assay samples.


Binding 4 a *


Chase


- -~








a complex with Hcfl06. tOE17 was cross-linked on the thylakoid membrane. AntiHcfl06 prevented the formation of cross-linking products. The crosslinking products were immunoprecipitated with antibody against Hefl06. These results suggest that Hcfl06 function in the precursor binding process, possibly as a receptor.



Discussion

For the first time, I show here that a typical Delta pH pathway precursor accumulated on the thylakoid membrane when the ApH was dissipated. The bound precursors were transported into the thylakoids when the ApH was restored. The construction of precursor tOE17 allows me to successfully demonstrate that transport on the Delta pH pathway occurs in two steps, binding and then translocation. I think that the binding step represents the targeting reaction on the Delta pH pathway. The tOE17 transit peptide only has N, H and C domains with the N domain containing MAGRR residues. Nevertheless, tOE17 is a typical substrate for the Delta pH pathway. Its transport does not require any soluble components from the chloroplast stroma and the ApH across the thylakoid membrane is the only energy requirement. Its transport also was competible with native precursor iOE23 from the Delta pH pathway (date not shown) and its transport was totally inhibited by Hcfl06 antibody. Of interest, the first precursor observed to exhibit productive binding is DT23. It has a dual targeting transit peptide but only can be transported by the Delta pH pathway (Henry et al. 1997). In the presence of ionophores DT23 transport was inhibited and substantial precursor remained bound to the thylakoid membrane. Because the DT transit peptide can target a cpSec pathway passenger protein transport on both Delta pH and cpSec pathways and can only target a Delta pH pathway








passenger on Delta pH pathway (Henry et al. 1997), it was thought that bound DT23 was stuck in the cpSec machinery. At that time no Delta pH pathway precursor had been found binding tightly to the thylakoid membrane. However, further experiment indicated that the bound DT23 was on the Delta pH pathway and was not stuck in Sec machinery (data not shown). Furthemore the bound DT23 was subsequently transported on the Delta pH pathway and competed by the Delta pH pathway precursor. All the experiments shown above for tOE17 were first tried with DT23 with similar results. We also found that native precursor iOE17 behaved the same in those experiments mentioned above, although the binding was much less. Why tOE17 can bind much tighter than the nature precursor on the thylakoid membrane is not clear. All of tightly bound precursors tOE17, DT23 and DT17 have a short transit peptide. However, tOE23 that also has a truncated (short) LTD similar to tOE17 does not bind strongly. The longer transit peptide in nature precursor has an A domain which function is unknown.

Precursor binding was sensitive to KCl concentration, indicating that an ionic interaction is involved. All Delta pH pathway precursor contain a twin-arginine motif which is crucial for targeting the precursor into the pathway (Chaddock et al. 1995). It is highly possible that the RR is used for precursor specific binding. However, without the RR, a truncated precursor named KK17 still bound on the thylakoid membrane, although it was not subsequently chased into the lumen (data not shown). This suggests that RR may not be involved in the binding and its role may be played at translocation step. Besides the twin-arginine, other domains in the transit peptide are also required for the targeting (Chaddock et al. 1995, Henry et al. 1997). Therefore, precursor binding could involve multiple components on the thylakoid membrane. Bound tOE17 could be cross-








linked to thylakoid proteins and the cross-linked complexes could be immunoprecipitated with Hcfl06 antibody. Comparing the cross-linking and immunoprecipitation products from bound tOE17 and KKl7 may give insight into whether or not RR specifically interacts with some components on the thylakoid membrane.

The binding competition tests in Figure 3-7 demonstrated that the binding was competible, implying that a saturable component of the transport machinery is involved in binding. However, when binding was conducted with increasing concentrations of 3Hlabeled E. coli-produced tOE 17 the total binding increased lineally with higher precursor usage. While the binding of E. coli-produced tOE17 is not satuable, the subsequent chase exhibited saturation. The direct transport also saturable and displayed the same kinetics (data not shown). The chase competition was unexpected. It is not likely but possible that under "chase conditions" the bound precursor is released into the solution and then reenters the transport pathway. If this is true, then transport of bound precursor cannot be considered to be chased from an intermediate step. The chase is considered to be a process in which the bound precursor directly transfers to the translocon from the binding site and is translocated into the lumen. In this case, the observed chase competition would actually be competition for a full transport reaction. Several methods, including conducting chase under different volumes of buffer, were tested to determine whether the chase was achieved directly. The results were inconclusive. However, we noticed that unlike the transport competition control a significant amount of bound precursor transported into the thylakoids regardless of how high the concentration of competitor was (Figure 3-8). This is evidence that the observed chase is different from the normal transport. The binding is considered to be an interaction between precursor and a








receptor. The subsequent chase should be directly achieved by shift of the bound precursor from the receptor to an associated translocon. Then, any competition occurring at this step must result from multiple receptors sharing a common translocon. Preliminary crosslinking data from Cline's laboratory (Mori and Cline, unpublished data) indicates that Hcfl06 exists in thylakoids as a multimer. If Hcfl06 functions as a receptor, competition for chase may reflect the existence of an HcflO6 multimer that serves as some sort of antenna receptor complex.

The Delta pH pathway is a very novel system considering that the ApH across thylakoids is the only energy requirement. It was originally thought to be a unique pathway of chloroplasts. Now its homologues have been found in bacteria (Settles and Martienssen 1998). Before this study, no Delta pH pathway precursors were found to bind tightly to the thylakoid membrane, making identification and purification of components very difficult. The binding-chase techniques developed in this study should provide an important tool to isolate machinery components and help understand the transport mechanism.













CHAPTER 4
SUMMARY AND CONCLUSIONS

The first part of my research is about protein transport on the cpSec pathway. Advancing the understanding about the mechanism is achieved by dissecting the transport process into targeting and translocation stages and the related characterization of each stage. Productive binding with precursors to the thylakoid membrane was stimulated with cpSecA and occurred when transport was inhibited by removing ATP from the assay with apyrase. cpSecA may function as a determinant for transport pathway selection as only the precursors using the cpSec pathway can form an complex with it. cpSecY was found to be another machinery component associated with bound precursor, providing the first evidence that cpSecY is functioning on cpSec transport pathway. Subsequent translocation of bound precursor required ATP, but was not stimulated by additional cpSecA. cpSecA had been speculated to be the Sec pathway receptor, given the fact that it is the only soluble component required for the transport on the cpSec pathway. It would be logical that cpSecA interacts with precursor in the stroma and targets the precursor to the translocon in the thylakoid membrane. However, no such interaction was detected. Our results support the notion that precursor commitment to the thylakoid cpSec pathway occurs on the membrane, rather than in the soluble stroma. Since the first detectable interaction between precursor and components of the cpSec pathway machinery was on the membrane, we assume that the receptor residences on the membrane.








The second part of my research is about protein transport on the Delta pH pathway. This pathway is unique for its special energy requirement and has been a recent focus of research in thylakoid protein transport. However, for years, knowledge about the mechanisms involved in this system has been quite limited. A biochemical approach such as cross-linking and native gel techniques to identify the machinery components had been difficult for the system since the routinely used precursors did not bind tightly to the thylakoid membrane. A newly constructed precursor protein tOE17 allows me to develop the technique of dissecting the whole transport into binding (targeting) and chase (translocation) steps. tOE17 binds to the membrane in substantial amount in the absence of the ApH and the bound precursor is chased into the lumen when the ApH is restored. Several observations suggest that binding is due to protein-protein interaction. Productive binding was competed by saturating amounts of a ApH pathway precursor protein, suggesting that machinery components from the Delta pH pathway are involved. Consisting with the transport requirement, the ApH was the only energy source used for chase. Interestingly, chase of the bound precursor could be competed with unlabeled precursor. Hcfl06 and Tha4 are two components identified recently. Both maize Hcf106 antibody and pea Tha4 antibody specifically inhibited tOE17 transport and chase to the pea thylakoids. However, only antibody raised against Hcfl06 reduced tOE17 binding to the thylakoid membranes. Given the fact that most part of the protein is facing the stroma, Hcfl06 may function as a receptor.














REFERENCES


Abad MS, Clark SE, Lamppa GK. 1989. Properties of a chloroplast enzyme that cleaves the chlorophyll a/b binding protein precursor. Plant Physiol. 90:117-24.

Akita M, Sasaki S, Matsuyama S, Mizushima S. 1990. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in E. coli. J. Biol. Chem. 265:8164-69.

Akiyama Y, Ito K. 1985. The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. EMBOJ. 4:3351-56.

Anderson CM, Gray J. 1991. Cleavage of the precursor of pea chloroplast cytochrome f by leader peptidase from Escherichia coli. FEBS Lett. 280:383-86.

Bassham DC, Bartling D, Mould RM, Dunbar B, Weisbeek P, Herrmann RG, Robinson C. 1991. Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins. J Biol Chem. 266:23606-10.

Berghbser J, Karnauchov I, Herrmann RG, Klosgen RB. 1995. Isolation and characterization of a cDNA encoding the secA protein from spinach chloroplasts. J. Biol. Chem. 270:18341-46.

Bogsch E, Brink S, Robinson C. 1997. Pathway specificity for a delta pH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein. EMBOJ. 16:3851-59.

Bogsch EG, Sargent F, Stanley NR, Berks BC, Robinson C, Palmer T. 1998. An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J. Biol. Chem. 273:18003-06.

Brundage L, Hendrick J, Schiebel E, Driessen A, Wickner W. 1990. The purified E. Coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell 62:649-657.

Caliebe A, Grimm R, Kaiser G, Ltibeck. J, Soll J, Heins L. 1997. The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein. EMBO J. 716:7342-7350.








Clark SA, Theg SM. 1997. A folded protein can be transported across the chloroplast envelope and thylakoid membranes. Mol. Biol. Cell 8:923-34.

Chaddock AM, Mant A, Karnauchov I, Brink S, Herrmann RG, Klosgen RB, Robinson C. 1995. A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta-pH-dependent thylakoidal protein translocase. EMBO J. 14:2715-22.

Chen X, Xu H, Tai PC. 1996. A significant fraction of functional SecA is permanently embedded in the membrane. SecA cycling on and off the membrane is not essential during protein translocation. J Biol. Chem. 271:29698-29706.

Chua N-H, Schmidt GW. 1978. Post-translational transport into intact chloroplasts of a precursor to the small subunit of riburose-1, 5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 75:6110-6114.

Chua N-H, Schmidt GW. 1979. Transport of proteins into mitochondria and chloroplasts. J. Cell Biol. 81:461-483.

Chitnis PR, Nechushtai R, Thornber JP. 1987. Insertion of the precursor of the lightharvesting chlorophyll a/b-protein into the thylakoids requires the presence of a developmentally regulated stromal factor. Plant Mol. Biol. 10:3-11.

Cline K, Werner-Washburne M, Lubben TH, Keegstra K. 1985. Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J. Biol. Chem. 260:3691-96.

Cline, K. 1986. Import of proteins into chloroplasts: Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates. J Biol. Chem. 261:1480414810[Abstract].

Cline K, Fulsom DR, Viitanen PV. 1989. An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited. J. Biol. Chem. 264:14225-32.

Cline K, Ettinger WF, Theg SM. 1992. Protein-specific energy requirements for protein transport across or into thylakoid membranes. J. Biol. Chem. 267:2688-96.

Cline K, Henry R, Li CJ, Yuan JG. 1993. Multiple pathways for protein transport into or across the thylakoid membrane. EMBO J 12:4105-14.

Cline K, Henry R. 1996. Import and routing of nucleus-encoded chloroplast proteins. Annu. Rev. Cell Dev. Biol. 12:1-26.

Creighton AM, Hulford A, Mant A, Robinson D, Robinson C. 1995. A monomeric, tightly folded stromal intermediate on the delta pH-dependent thylakoid protein transport pathway. J. Biol. Chem. 270:1663-69.










Cunningham K, Lill R, Crooke E, Rice M, Moore K, Wickner W, Oliver D. 1989. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 8:955-959.

de Boer AD, Weisbeek PJ. 1991. Chloroplast protein topogenesis: import, sorting and assembly. Biochim. Biophys. Acta 1071:221-53.

Dobberstein B, Blobel G, Chua, N-H. 1977. In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 74:1082-1085.

Duong F, Wickner W. 1997. Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme. EMBO J 16:2756-68.

Economou A, Wickner W. 1994. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell 78:835-843.

Ento T, Kawakami M, Gogo A, America T, Weisbeek P, NaKai M. 1994. Chloroplast protein import: chloroplast envelopes and thylakoids have different abilities to unfold proteins. Eur. J. Biochem. 225:403-409.

Fikes JD, Bassford PJ Jr. 1989. Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide. JBacteriol. 171:402-409.

Fincher V, McCaffery M, Cline K. 1998. Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway. FEBS Letters 423:66-70.

Friedman AL, Keegstra K. 1989. Chloroplast protein import: quantitative analysis of precursor binding. Plant Physiol. 89:993-99.

Haehnel W, Jansen T, Gause K, Klbsgen RB, Stahl B, et al. 1994. Electron transfer from plastocyanin to photosystem I. EMBO J. 1:1028-38.

Hageman J, Baecke C, Ebskamp M, Pilon R, Smeekens S, Weisbeek P. 1990. Protein import into and sorting inside the chloroplast are independent processes. Plant Cell 2:479-94.

Halpin C, Elderfield PD, James HE, Zimmerman R, Dunbar B, Robinson C. 1989. The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical EMBO J. 8:3917-21.

Hand JM, Szabo LJ, Vasconcelos AC, Cashmore AR. 1989. The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence. EMBOJ 8:3195-3206.








Hartl F, Lecker S, Schiebel E, Hendrick J, Wickner W. 1990. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. Coli plasma membrane. Cell 63:269-279.

Haward SR, Napier JA, Gray JC. 1997. Chloroplast SecA functions as a membraneassociated component of the Sec-like protein translocase of pea chloroplasts. European Journal of Biochemistry. 248:724-730.

Henry R, Carrigan M, McCaffery M, Ma XY, Cline K. 1997. Targeting determinants and proposed evolutionary basis for the Sec and delta-pH protein transport systems in chloroplast thylakoid membranes. J. Cell Biol. 136:823-32.

Hinnah SC, Hill K, Wagner R, Schlicher T, Soil J. 1997. Reconstitution of a chloroplast protein import channel. EMBO J. 16:7351-7360.

Hirsch S, Muckel E, Heemeyer F, von Heijne G, Sol J. 1994. A receptor component of the chloroplast protein translocation machinery. Science 266:1989-92.

Hoffman NE, Franklin AE. 1994. Evidence for a stromal GTP requirement for the integration of a chlorophyll a/b-binding polypeptide into thylakoid membranes. Plant Physiol. 105:295-304.

Hulford A, Hazell L, Mould RM, Robinson C. 1994. Two distinct mechanisms for the translocation of proteins across the thylakoid membrane, one requiring the presence of a stromal protein factor and nucleotide triphosphates. J. Biol. Chem. 269:3251-56.

Hynds PJ, Robinson D, Robinson C. 1998. The Sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane. J Biol. Chem. 273:34868-34874.

Keegstra K, Cline K. 1999. Protein import and routing systems of chloroplasts. Plant Cell. In press.

Kessler F, Blobel G, Patel HA, Schnell DJ. 1994. Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science 266:1035-39.

Kessler F, Blobel G. 1996. Interaction of the protein import and folding machineries in the chloroplast. Proc. Natl. Acad Sci. USA 93:7684-89.

Kim SJ, Robinson D, Robinson C. 1996. An Arabidopsis thaliana cDNA encoding PS IIX, a 4.1 kDa component of photosystem II: a bipartite presequence mediates SecA/delta pH-independent targeting into thylakoids. FEBS Lett. 390:175-78.

Kirwin PM, Meadows JW, Shackleton JB, Musgrove JE, Elderfield PD, et al. 1989. ATPdependent import of a lumenal protein by isolated thylakoid vesicles. EMBO J 8:225155.









Klimyuk, VI, Persello-Cartieaux F, Havaux M, Contard-David P, Schuenemann D, Meiherhoff K, Gouet P, Jones JDG, Hoffman NE, Nussaume L. 1999. A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting. Plant Cell, 11:87-99.

Knott TG, Robinson C. 1994. The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane. J. Biol. Chem. 26911:7843-46.

Ko K, Cashmore AR. 1989. Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein. EMBO J. 8:3187-94.

Kouranov A, Schnell DJ. 1997: Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts. J. Cell Biol. 139: 1677-1685.

Kuhn A, Kiefer D, K6hne C, Zhu H-Y, Tschantz WR, Dalbey RE. 1994. Evidence for a loop-like insertion mechanism of pro-Omp A into the inner membrane of Escherichia coli. Eur. J. Biochem. 226:891-897.

Laidler V, Chaddock AM, Knott TG, Walker D, Robinson C. 1995. A SecY homolog in Arabadopsis thaliana. JBiol. Chem. 270:17664-17667.

Lamppa GK. 1988. The Chlorophyll a/b-binding protein inserts into the thylakoids independent of its cognate transit peptide. J. Biol. Chem. 263:14996-14999.

Lorkovic ZJ, Schraser WP, Pakrasi HB, Irrgang KD, Herrmann RG, Oelmiller R. 1995. Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach. Proc. Natl. Acad Sci. USA 92:8930-34.

Ma YK, Kouranov A, LaSala SE, Schnell DJ. 1996. Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. J. Cell Biol. 134:315-27.

Meadows JW, Robinson C. 1991. The full precursor of the 33-kDa oxygen-evolving complex protein of wheat is exported by Escherichia col and processed to the mature size. Plant Mol. Biol. 17:1241-43.

Meyer T H, Mnr6tret J F, Breitling R, Miller K R, Akey CW, Rapoport T A. 1999. The bacterial SecY/E translocation complex forms channel-like structures similar to those of the eukaryotic Sec61p complex. J Mol. Biol. 285:1789-800.








Michl D, Robinson C, Shackleton JB, Herrmann RG, K16sgen RB. 1994. Targeting of proteins to the thylakoids by bipartite presequences: CFoll is imported by a novel, third pathway. EMBO J. 13:1310-17.

Mizushima S, Tokuda H. 1990. In vitro translocation of bacterial secretory proteins and energy requirements. J. Bioenerg. Biomembr. 22:389-99. Review.

Mori H, Cline K. 1998. A signal peptide that directs non-See transport in bacteria also directs efficient and exclusive transport on the thylakoid Delta pH pathway. J. Biol. Chem. 273:11405-11408.

Mori H, Summer L, Ma X, Cline K. 1999. Component specificity for the thylakoidal See and Delta pH-dependent protein transport pathways. (To be submitted).

Morin XK, Soil J. 1997. Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery. Planta 201:119-127.

Nilsson R, Brunner J, Hoffman NE, van Wijk KJ. 1999. Interactions of ribosome nascent chain complexes of the chloroplast-encoded Dl thylakoid membrane protein with cpSRP54 EMBO J 18:733-742.

Nishiyama K-I, Fukuda A, Morita K, Tokuda H. 1999. Membrane deinsertion of SecA underlying proton motive force-dependent stimolation of protein translocation. EMBO J. 18:1049-1058.

Nohara T, Asai T, Nakai M, Sugiura M, Endo T. 1996. Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway. Biochem. Biophys. Res. Commun. 224:474-78.

Oliver DB, Cabelli RJ, Dolan KM, Jarosik GP. 1990. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc. Natl. Acad. Sci. USA 87:8227-31.

Olsen LJ, Keegstra K. 1992. The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J. Biol. Chem. 267:433-39.

Osborne RS, Silhavy TJ. 1993. PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J 12:3391-3398.

Payan LA, Cline K. 1991. A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein. J. Cell Biol. 112: 603-13.

Pain D, Blobel G. 1987. Protein import in chloroplasts requires a chloroplast ATPase. Proc. Natl. Acad. Sci. USA 84:3288-92.








Perry SE, Keegstra K. 1994. Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell 6:93-105.

Pilon M, De Boer AD, Knols SL, Koppelman MHGM, Van der Graaf RM, De Kruijff B, Weisbeek PJ. 1990. Expression in Escherichia coli and purification of a translocationcompetent precursor of the chloroplast protein ferredoxin J. Biol. Chem. 265:3358-3361.

Pugsley A.1993. The complete general secretory pathway in gram-negative bacteria Microbiol Rev. 57:50-108.

Puziss JW, Fikes JD, Bassford PJ Jr. 1989. A analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide. J Bacteriol. 171:23022311.

Rapoport TA, Jungnickel B, Kutay U. 1996. Protein Transport Across the Eukaryotic Endoplasmic Reticulum and Bacterial Inner Membranes Annu. Rev. Biochem. 65:271-303.

Robinson C, Ellis RJ. 1984. Transport of proteins into chloroplasts: partial purification of a chloroplast protease involved in the processing of imported precursor polypeptides. Eur. J. Biochem. 142:337-42.

Robinson C, Kl6sgen RB. 1994. Targeting of proteins into and across the thylakoid membrane: a multitude of mechanisms. Plant Mol. Biol. 26:15-24.

Robinson D, Karnauchov I, Herrmann RG, Kl6sgen RB, Robinson C. 1996. Proteasesensitive thylakoidal import machinery for the Sec-, delta pH- and signal recognition particle-dependent protein targeting pathways, but not for CFoI integration. Plant J 10:149-55.

Robinson C, Hynds PJ, Robinson D, Mant A. 1998. Multiple pathways for the targeting of thylakoid proteins in chloroplasts. Plant Molecular Biology 38:209-221.

Sargent F, Bogsch EG, Stanley NR, Wexler M, Robinson C, Berks BC, Palmer T. 1998. Overlapping functions of components of a bacterial Sec-independent protein export pathway EMBO J1 17:3640-3650.

Shaw AS, Rottier PJIM, Rose JIK. 1988. Evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 85:7592-7596.

Schiebel E, Driessen AJM, Hard F-U, Wickner W. 1991. AplO and ATP function at different Steps of the catalytic cycle of preprotein translocase. Cell 64:927-939.

Schnell DJ, Blobel G. 1993. Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelop contact sites. J Cell Biol. 120:103-15.








Schnell DJ, Kessler F, Blobel G. 1994. Isolation of components of the chloroplast protein import machinery. Science 266:1007-12.

Schnell DJ. 1998. Protein targeting to the thylakoid membrane. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:97-126.

Schuenemann D, Gupta S, Persello-Cartieaux F, Klimyuk VI, Jones JDG, Nussaume L, Hoffman NE. 1998. A novel signal recognition particle targets light harvesting proteins to the thylakoid membranes. Proc. Natl. Acad. Sci. USA 95:10312-10316.

Settles AM, Yonetani A, Baron A, Bush DR, Cline K, Martienssen R. 1997. Secindependent protein translocation by the Maize Hefl06 Protein. Science 278:1467-1470

Settles AM, Martienssen R. 1998. Old and new pathways of protein export in chloroplasts and bacteria. Trends Cell Biol. 8:494-501. Review.

Seidler A, Michel H. 1990. Expression in Escherichia coli of the psbO gene encoding the 33 kD protein of the oxygen-evolving complex from spinach. 'EMBO . 9:1743-48.

Smeekens S, Bauerle C, Hageman J, Keegstra K, Weisbeek P. 1986. The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell. 46:365-75.

Soil J, Tien R. 1998. Protein translocation into and across the chloroplastic envelop membranes. Plant Molecular Biology 38:191-207.

Sugiura M, Hirose T, Sugita M. 1998. Evolution and mechanism of translation in chloroplasts. Annu. Rev. Genet. 32:437-459.

Swidersky UE et al. 1992. Biochemical analysis of the biogenesis and function of the Escherichia col export factor SecY. Eur. J. Biochem. 207:803-811.

Swidersky UE, Hoffschulte HK, Muller M. 1990. Determinants of membrane-targeting and transmembrane translocation during bacterial protein export. EMBO J 9:1777-1785.

Theg SM, Bauerle C, Olsen LJ, Selman BR, Keegstra K. 1989. Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem. 264:6730-36.

Tranel PJ, Froeblich J, Goyal A, Keegstra K. 1995. A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway. EMBOJ 14:2436-46.

VanderVere PS, Bennett TM, Oblong JE, Lamppa GK. 1995. A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases. Proc. Natl. Acad Sci. USA 92:7177-81.









Viitanen PV, Doran ER, Dunsmuir P. 1988. What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein? J Biol. Chem. 263: 15000-15007.

Voelker R, Barkan A. 1995. Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid. EMBO J 14:3905-14.

Voelker R, Mendel-Hartvig J, Barkan A. 1997. Transposon-disruption of a maize nuclear gene, thal, encoding a chloroplast SecA homologue: in vivo role of cp-SecA in thylakoid protein targeting. Genetics 145:467-78.

von Heijne G, Steppuhn J, Herrmann RG. 1989. Domain structure of mitochondrial and chloroplast targeting peptides. Eur. J Biochem. 180:535-45.

von Heijne G, Nishikawa K. 1991. Chloroplast transit peptides: the perfect random coil? FEBS Lett. 278(1):1-3

Waegemann K, Paulsen H, Soil J. 1990. Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence. FEBS Lett. 261:89-92.

Watanabe P, Blobel G. 1989. Site-specific antibodies against the PrlA (SecY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preproteins. Proc NatlAcad Sci USA 86:1895-1899.

Weisbeek P, Hageman J, De Boer D, Pilon R, Smeekens S. 1989. Import of proteins into the chloroplast lumen. J. Cell Sci. Suppl. 11: 199-223.

Weiner JH, Bilous PT, Shaw GM, Lubitz SP, Frost L, Thomas GH, Cole JA, Turner RJ. 1998. A novel and ubiquitous system for membrane targeting and secretion of cofactorcontaining proteins. Cell 93:93-101.

Wexler Margaret, Bogsch Erik G, Kl6sgen Ralf Bemd, Palmer Tracy, Robinson Colin, Berks Ben C. 1998. Targeting signals for a bacterial Sec-independent export system direct plant thylakoid import by the ApH pathway, Febs Letters 431:339-342.

Yuan J, Cline K. 1994a. Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity. J. Biol. Chem. 269:18463-67.

Yuan J, Henry R, McCaffery M, Cline K. 1994b. SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting. Science 266:796-98.

Yuan J, Henry R, Cline K. 1993. Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70. Proc. Natl. Acad. Sci. USA 90:8552-56.





94



Zak E, Sokolenko A, Unterholzner G, Altschmied L, Herrmann RG. 1997. On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes. Planta 201:334-41.




Full Text

PAGE 1

PROTEIN TARGETING AND TRANSLOCATION ON cpSEC AND DELTA pH PATHWAYS IN CHLOROPLAST THYLAKOIDS By XIANYUE MA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1999

PAGE 2

ACKNOWLEDGMENTS The author thanks his supervisor, Dr. Kenneth Cline, for providing an exceptional environment where the author received full training in both research skills and scientific thinking. The author appreciates the advice and care from the members of his committee Drs. Kenneth Cline, Alice Harmon, Alfred S. Lewin, Daryl R. Pring, and Carlos Eduardo Vallejos. The author especially thanks Dr. Ralph Henry for his kind help in the author's experimental operations. Dr. Henry's early work with DT23 also helped the author's research project. The author wants to thank Dr. Baocai Tan and Wentao Deng for their help in DNA work such as tOE17 subcloning and expression. Thanks go to Dr. Hiroki Mori for providing antibodies. The author also wishes to thank Mike McCafifery, Vivian Fincher, Shaw Wu, and Dr. Liz Summer for their help. This dissertation is dedicated to the author's 81 -year old mother Yisong Wu for asking her forgiveness for her son's years of absence. ti

PAGE 3

TABLE OF CONTENTS page ACKNOWLEDGMENTS ii KEY TO ABBREVIATIONS iv ABSTRACT v CHAPTERS 1 LITERATURE REVIEW 1 Protein Import into Chloroplast Stroma 2 Protein Transport into Thyiakoids 5 Summary and Perspective 18 2 PROTEIN TARGETING AND TRANSLOCATION ON THE cpSec PATHWAY 22 Abstract 22 Introduction 23 Materials and Methods 25 Results 29 Discussion 46 3 TARGETING AND TRANSLOCATION ON THE DELTA pH PATHWAY 54 Abstract 54 Introduction 55 Materials and Methods 56 Results 59 Discussion 79 4 SUMMARY AND CONCLUSIONS 83 REFERENCES 85 BIOGRAPHICAL SKETCH 95 iii

PAGE 4

. KEY TO ABBREVIATIONS APDP N-r4-(p-Azidosalicylamido) butyl]-3'-r2'-pyridyldithio] propionamide BS^ bis [sulfosuccinimidyl] suberate CCCP carbonylcyanide 3 -chlorophenylhydrazone DSS disuccinimidyl suberate DTT dithiothreitol LTD lumen targeting domain LHCP light-harvesting chlorophyll a/b protein MBS m-Maleimidobenzoyl-N-hydroxysuccinimide ester 0E17 the 1 7-kDa subunit of the oxygen-evolving complex OE23 the 23-kDa subunit of the oxygen-evolving complex OE33 the 33-kDa subunit of the oxygen-evolving complex Pmf proton motive force PC plastocyanin SE stromal extract STD stroma targeting domain iv

PAGE 5

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PROTEIN TARGETING AND TRANSLOCATION ON cpSEC AND DELTA pH PATHWAYS IN CHLOROPLAST THYLAKOIDS By XIANYUEMA May 1999 Chair: Kenneth C. Cline Major Program: Plant Molecular and Cellular Biology Protein transport is required for biogenesis of the chloroplast, the site of photosynthesis. There are at least two pathways for protein transport into the chloroplast thylakoid lumen. One pathway is named the cpSec pathway, another is named the Delta pH pathway. Each pathway is specific for a subset of precursor proteins. I hypothesize that targeting, a specific interaction between the precursor and components of the protein transport machinery, commits precursor proteins to a specific pathway. I have attempted to describe and characterize the first committed step on the cpSec pathway and to defme in general terms the sequences required for commitment. Binding of precursors to the thylakoid membrane appears to be the first committed step. It required stromal protein, of which cpSecA was the essential component, and occurred when transport was inhibited by removing ATP from the assay with apyrase. Subsequent transport of bound precursor required ATP, but was not stimulated by additional cpSecA.

PAGE 6

My results show that precursor binding to the membrane results in formation of a large complex that includes precursor, cpSecA, and cpSecY. The complex formation with cpSecA is precursor specific, requiring a cpSec transit peptide and additionally a cpSeccompatible flanking region. Techniques were developed to dissect protein transport on the Delta pH pathway into binding (targeting) and chase (translocation). A newly constructed precursor protein tOE17 that uses the Delta pH pathway, bound to the membrane in the absence of the ApH. Binding is productive because bound precursor was transported into the lumen when the ApH was restored. Productive binding was competed by saturating amounts of a Delta pH pathway precursor protein, suggesting that precursor tOE17 binds to components of the Delta pH pathway translocation machinery. Consistent with the transport requirement, the ApH was the only energy source used for chase. Interestingly, the chase of bound precursor was also competed by over expressed precursor. Antibodies raised to E. co//-expressed Hcfl06 specifically inhibited tOE17 transport and reduced the level of productive binding. My results suggest that Hcfl06 functions in early steps of the transport process, possibly as a receptor. vi

PAGE 7

CHAPTER 1 LITERATURE REVIEW Although the sun is the primary energy source for all life, it is in chloroplast that the light energy from the sun is converted into biologically usable chemical energy. Apart from photosynthesis, several other important biosynthetic processes also occur in chloroplasts, such as biosynthesis of amino acids, lipids, pigments and plant hormones. A mature chloroplast can be spatially divided into six compartments: outer envelope, inner envelope, interenvelope space, stroma, thylakoid membrane and thylakoid lumen. The chloroplast is enclosed by the envelopes; inside the chloroplast the thylakoid stacks float in the stroma. The well differentiated chloroplast develops from a small, more or less undifferentiated proplastid. This morphogenetic process is controlled by endogenous and exogenous factors and involves a massive production of proteins that are required for the fimctions of the chloroplast. The chloroplast is a semi-autonomous organelle. It contams genetic material and has functional systems for both gene transcription and translation. However, its circular DNA molecule contains only about 100 genes, about 69-77 of which encode proteins (Sugiura et al. 1998). More than three fourth of proteins residing in the chloroplasts are nucleus encoded and synthesized in the cytoplasm. These proteins must be imported into the chloroplast from the cytosol. It is obvious that protein import plays an important role in chloroplast biogenesis. 1

PAGE 8

Protein Import into Chloroplast Stroma Significant advances in understanding chloroplast protein transport began two decades ago when protein import was reconstituted with isolated chloroplasts (Chua and Schmidt 1978, 1979). The in vitro or in organella transport assay is conducted by mixing radiolabeled precursor protein with purified chloroplasts. Active chloroplasts for protein import are prepared by homogenization of fresh young green tissues followed by Percoll gradient centrifiigation and isotonic buffer wash. Radiolabeled precursor proteins are prepared by in vitro translation of mRNA with a wheat germ system (Cline et al. 1989) or rabbit reticulocyte system in the presence of one radiolabeled amino acid. Large amounts of precursor protein can be made by expression of the protein in E. coli and subsequent purification (Weisbeek et al. 1989; Pilon et al. 1990; Waegemann 1990) Stroma Targeting Domain A nuclear-encoded chloroplast protein is initially synthesized in the cytosol as a precursor protein that contains a transient transit peptide at its amino terminus (Dobberstein et al. 1977). For stroma-targeted precursor the transit peptide consists of a stromal targeting domain (STD) that governs the import of the precursor across the chloroplast envelope. Once in stroma the precursor stromal targeting domain is cleaved by a processing protease (Robinson and Ellis 1984, Abad et al. 1989 VanderVere et al. 1995). Stromal targeting domains range from 30 to 120 amino acid residues and are predicted to have a random coil conformation (von Heijne and Nishikawa 1991). Although the stromal targeting domains from different precursors are interchangeable for

PAGE 9

3 targeting passenger proteins across the chloroplast envelope, there are no conserved sequence blocks among the stromal targeting domains. However, stromal targeting domains are characterized to share three compositional motifs: a 10-15 residue Nproximal portion without charged residues, glycine or proline; a variable middle region that lacks negative charged residues and is rich in hydroxylated amino acids (serine and threonine); and a C-terminal region that contains a loosely conserved sequence (IleA'alX-Ala/Cys*Ala) for proteolytic processing (von Heijne et al. 1989, de Boer and Weisbeek 1991) The Import Process Protein import across chloroplast envelope into the stroma is considered to be a three step process (Cline and Henry 1996, Schnell 1998, Soli and Tien 1998). The first is reversible precursor binding to the outer envelope. This step does not require any energy. The binding is achieved via the interaction of the precursor stromal targeting domain with proteuiaceous receptors and possibly polar lipids at the surface of outer membrane (Cline et al. 1985). The translocon at the outer chloroplastic envelope membrane (Toe complex) contains three integral membrane proteins: Toc34, Toc86 and Toc75. Two lines of evidences suggest that Toc86 and Toc34 function as primary receptors: first, crosslinking studies indicate that Toc86 and Toc34 are contact with the precursor during the initial binding (Perry and Keegstra 1994, Ma et al. 1996, Kouranov and Schnell 1997); second, anti-Toc86 IgGs block the binding of precursor to the envelope (Hirsch et al. 1994).

PAGE 10

4 In the second step of import, the precursor inserts across the outer envelope, resulting in close physical proximity to Toc75 (Perry and Keegstra 1994, Ma et al. 1996). Toc75 is deeply embedded in the outer envelope (Schnell et al. 1994, Tranel et al. 1995). Analysis of the Toc75 sequence indicates that it may traverse the membrane with 16 hydrophobic P'Strands. Toc75 is therefore proposed to function as a protein translocation channel and this was verified in vitro by studies using expressed Toc75 reconstituted into liposomes (Hirmah et al. 1997). Unlike the first step of import in which the binding is energy indep>endent and reversible, the second step requires the hydrolysis of both ATP and GTP (less than 100 ^M) and results a tight association of the precursor with the Toe complex (Kessler et al. 1994, Olsen and Keegstra 1992, Ma et al. 1996). Precursor insertion across the outer envelope also brings the transit peptide (stromal targeting domain) near to the translocon at the inner chloroplastic envelope membrane (Tic complex) (Schnell and Blobel 1993). In their in vitro binding assay, Friedman and Keegstra (1989) and Schnell and Blobel (1993) estimated that there are about 1500-3000 import sites per chloroplast. However, immunogold labeling of Toc34 in cryo substituted thin sectioned chloroplasts suggested that there were at least 15000-20000 import sites (Morin and Soil 1997). The difference indicates that in the isolated chloroplasts only a small percentage of import sites are functional for precursor binding or import, implying that only receptors in a certain state can be used. Both Toc86 and Toc34 are GTP-binding proteins (Kessler et al, 1 994). It is possible that GTP status may determine receptor state. Several proteins have been identified as components of the translocon at the iimer chloroplastic envelope. They are Tic 14, Tic21, Tic22, Tic55, TicllO etc. Tic 14, Tic21 and Tic22 were identified by cross-linking to a precursor protein (Ma et al 1996,

PAGE 11

5 Kouranov and Schnell 1997). Tic22 is peripherally localized at the out face of the inner envelope and considered to be an inner envelope receptor. TicllO was found associated with two stroma chaperones, cpn60 and ClpC; it may be involved in driving precursor transport or in folding of newly imported proteins in the stroma (Kessler and Blobel 1996). Tic55 was found associated with TicllO and the Toe components Toc86, Toc34 and Toc75 in both blue-native gel electrophoresis and affinity chromatography assays (Cahebe et al. 1997). Tic55 contains an iron sulfur cofactor. Soil and Tien (1998) hypothesized that Tic55 may act as a regulatory factor by using the iron-sulfur cluster as a redox sensor to influence protein transport. The third step of import is protein translocation into the stroma. This step requires the cooperation of both Toe and Tic complexes. The precursor is transported across both outer and inner envelopes via the contact sites (Schnell and Blobel, 1993). ATP hydrolysis at higher concentration (>100 |^M) in the stroma is used as energy source (Pain and Blobel 1987, Theg et al. 1989). The stromal targeting domain is cleaved by a soluble processing peptidase during or immediately following protein import into the stroma, creating a mature stromal protein (Robinson and Ellis 1984, Abad et al. 1989, VanderVereetal. 1995). Protein Transport into Thylakoids Proteins destined for the thylakoid lumen have a bipartite transit peptide: the aminoproximal region is a stroma-targeting domain (STD); the STD is followed by a lumentargeting domain (LTD) (Ko and Cashmore 1989, Hageman et al. 1990). The bipartite nature of the transit peptide was first proposed by Smeekens et al. (1986) based on their

PAGE 12

6 assay with chimeric precursor proteins. The precursor PC transit peptide not only directed the precursor into the chloroplast stroma but also targeted the mature PC into its subsequent localization. Import of the precursor across the chloroplast envelope is governed by the STD of the transit peptide and the import process is the same as described above for a nucleus-encoded stromal protein. The STD is cleaved by a processing protease in the stroma, exposing the LTD and giving rise to an intermediate precursor protein. The LTD fiirther directs transport into the thylakoid lumen where the LTD is removed by a second processing protease, thereby producing the mature protein (Halpinetal. 1989). In vivo and in organella, the precursor imports across the chloroplast envelope first and then transports into the thylakoid lumen. However, import and transport are independent processes. With in vitro assays, precursor can be directly transported into the thylakoids bypassing the import step (Cline 1986, Kirwin et al. 1989). A stromal intermediate precursor was accumulated when thylakoid transport was inhibited during a chloroplast import assay (Cline et al. 1989). The intermediate was in a productive state and it could be chased into the thylakoids once the transport inhibition was removed (Cline et al. 1993). The plastid-encoded thylakoid protein cytochrome /is synthesized with only a LTD, its transport should not have any relation with the import process (Nohara et al. 1996, Zak et al. 1997). Actually, full-length precursor with both STD and LTD are efficient substrates for transport into isolated thylakoids (Robinson and Klosgen 1994). This impUes that thylakoid protein transport employs a loop mechanism similar to that used by the E. coli Sec system and the endoplasmic reticulum transport system (Shaw et al. 1988, Kuhn et

PAGE 13

al. 1994). This hypothesis was clearly proven by Fincher et al. (1998). The topology of precursor insertion into the thylakoid membrane was investigated with a fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17. While the mature 0E17 was transported to the thylakoid lumen, the amino terminus, including the pOE17 transit peptide, remamed on the cis side of the thylakoid membrane. Although there appears to be one major pathway for protein import across the chloroplast envelope, there are at least four pathways for protein transport into the thylakoid lumen or integration in the thylakoid membrane (Figure 1-1). Multiple pathways for thylakoid protein transport were first recognized by the discovery that different subgroups of precursors had different energy and soluble protein factor requirements for translocation (Cline et al. 1992). The existence of pathway specific translocation machinery was further demonstrated by precursor competition studies (Cline et al. 1 993). For example, saturating concentration of iOE23 inhibited transport of OEl 7, OE23 and PSHT, but was without effect on transport of OE33 and PC. One of the thylakoid transport pathways is called the cpSec pathway since this pathway employs a chloroplast homologue of the bacterial SecA protein. A second pathway is named the Delta pH pathway because in this pathway, the ApH across the thylakoid membrane is the only energy requirement for protein transport. The third is the cpSRP pathway and the fourth is the spontaneous pathway. Each pathway is specific for a subset of precursor proteins (Cline and Henry 1996, Schnell 1998, Robinson et al. 1998). In this dissertation, research focuses on protein transport on the cpSec and Delta pH pathways. Literature about protein transport on these two pathways will be reviewed in the following sections.

PAGE 14

8 pre-tumenal P'""'^'' protein I pre-membrane protein cpSRP TftCf I iOE23 iOE17 iPS2T IPS1N GTP+ A pH stim. outer envelope Inner envelope troma C10II Ps2W Ps2X lumen thylakold membrane Figure 1-1. Schematic representation of the one general pathway for protein import across chloroplast envelope and four known pathways for protein transport to the thylakoids (From Keegstra and Cline, 1999).

PAGE 15

9 The LTD Structure and Precursor Targeting The LTD can be divided into four sub-domains based on the nature of the amino acid residues (Cline and Henry 1996): an acidic (A) domain of about 12-15 amino acid residues is on the amino-terminus of the LTD; this is followed by a positively charged (N) domain of about 4-7 amino acid residues; then a hydrophobic (H) domain of 12-18 amino acid; and a C-terminal (C) domain with a relatively conserved motif of Ala-X-Ala for proteolytic processing. Figure 1-2 shows some examples of LTDs that confer either cpSec or Delta pH pathway specifically. Also shown is an artificially produced LTD that confers transport on both pathways. The N, H and C domains of LTD represent the basic structure of the classical signal peptides that direct proteins to the endoplasmic reticuliun or the bacterial plasma membrane. This implies that the LTD is evolutionarily related to these signal peptides and that thylakoid protein transport is conserved from the original bacterial endosymbiont (von Heijne et al. 1989). Additional support for the conservative origin of LTDs came from the feet that LTD is cleaved by a thylakoid lumenal processing peptidase (TPP) with identical specificity to a bacterial leader peptidase (LPP), which can carry out feithful processing if the protein is expressed in E. coli and exported across bacterial inner membrane (Halpin et al. 1989, Anderson et al. 1991). Thylakoid proteins OE33 and plastocyanin, as well as DT23 with a dual targeting transit peptides, were exported to the periplasmic space and properly processed to their mature sizes when e}q)ressed in E. coli (Seidler and Michel 1990, Meadows and Robinson 1991, Haehnel et al. 1994, Henry et al. 1997). Also, Mori and CUne (1998), and Wexler et al. (1998) reported that the signal

PAGE 16

10 N H + --H+ Delta pH AOKODDWDAWSR RLALSVLIGAAAV GSKVSPADA Pathway . . ++ + ASAEGDAVAOAGRR AVIGLVATGIVGGAL SQAARA + + ++ MPVIKAORWGDDVDGSNGRR SAMVFLAATLFSTAAV SASANA -++ ++ AOVESVOMSGERKTEGNNGRRE MMFAAAAAAICSVAGV ATA Sec Pathway + SSLK DFGVAIAVATAASIVLA GNAMA + Precursors OE23 OE17 PSIN PSIIT PC OE33 + -+.+.+ AFGLEHYGAKVTCSLQSDFKELAHKCVEASK L\GFALATSALW SGASA + +--+++ APRSKIVCQQENDQQQPKKELAKVGAN AAAALALSSVLLSSWSV APDAAMA PSIF Dual-Pathway MVSRRFGVLWATAASIVLAGNAMA DT23, DT33 Figure 1-2. Lumen targeting domain (LTD) structure of precursors targeted to the cpSec, the Delta pH pathway, or to both pathways. The acidic (A), charged (N), hydrophobic (H) and cleavage (C) regions of LTD are shown. A hybrid dual targeting transit peptides (DT) is composed of the OE23 N region fused to the PC H region. The hydrophobic amino acids that comprise the H domain are underlined whereas charged amino acids are indicated with (+) or (-). The sequences of the LTD for OE23, OE33 and PC correspond to the amino terminus determined by Bassham et al (1991). Precursors: PC (plastocyanin from Arabidopsis); 0E17, OE23, OE33 (17, 23 and 33-kD proteins of the oxygen-evolving conqilex from maize, pea and pea); PSIN i[OTa. Arabidopsis; PSIIT from cotton; PSIF from spinach.

PAGE 17

11 peptide from an E. coli precursor could direct thylakoid passenger protein into the thylakoids. The general structures of LTDs for both cpSec and Delta pH pathways are the same. With a compatible passenger, N, H and C domains in the LTD are sufficient for precursor targeting (Henry et al. 1994, 1997). The function of the A domain is currently unclear. Certain sub-domains of LTD determinate pathway specificity. For the Delta pH pathway, the N domain contains the specificity element. All the Delta pH pathway precursors contain a double arginine (RR) motif in the N domain, which is essential for the Delta pH pathway transport (Chaddock et al. 1995, Henry et al. 1997). It is likely that a receptor would interact with the double arginines, possibly combined with the non specific hydrophobic core, and bring the precursor to the Delta pH pathway translocation machinery. In contrast to the Delta pH pathway, the H domain requirement is more specific for cpSec pathway precursor targeting, while the N domain requirement is non specific (Henry et al. 1997). So, a different receptor could interact with the H domain and target the precursor to the cpSec pathway. The "domain determinant" hypothesis was fiirther examined with artificial precursor DT-PC, which contains determinants for both pathways. DT-PC was constructed by replacing the N domain of cpSec pathway precursor PC with one from Delta pH pathway precursor OE23. This precursor incredibly can be efficiently transported on both pathways (Henry et al. 1997). Although "domam determinant elements" are key factors for pathway specificity, they alone seem insufficient for targetmg. For example, amino acid residues surrounding the RR motif are important for the Delta pH pathway targeting (Bogsch et al. 1997).

PAGE 18

12 In addition, results from Henry et al. (1994, 1997) and Clausmeyer et aL (1993) indicate that the mature protein plays a role in targeting and/or translocation on the cpSec pathway. For example, although a cpSec LTD could direct the Delta pH pathway passenger 0E17 transport on the cpSec pathway, the transport was inefficient. Secondly, 'i the hybrid (DT) transit peptide shown in Figure 1-2, while directing efficient transport of cpSec passengers on both pathways, was not able to direct efficient transport of the Delta pH passengers on the CpSec pathway. In E. coli secretory proteins, a 30-residue-long region immediately downstream of the signal peptides has been termed the "export initiation domain". Introducing positively charged amino acid residues mto the domain inhibits E. coli protein export at an early stage (Anderson & van Heijine 1991, 1993; Yoshihisa & Ito 1996). The inhibition could be reversed by adding negatively charged amino acids around the positively charged ones (Anderson & van Heijine 1991). Comparing the sequences of different precursors of the thylakoid cpSec pathway, we have observed that a 10-40 residue long region (adjacent to the LTD in PC and OE33, more distant from the LTD in PSIF) is characterized as having net zero or negative charge and containing a pair of special amino acid residues (NN or NQ, N-asparagine, Q-glutamine). This region may be analogous to the "export initiation domain". I speculate that it is this region that may be involved in the interaction between the precursor mature sequence and cpSec pathway transport machinery. The Delta pH pathway precursors generally have a net positive charge in this region and lack NN or NQ. This may be the reason why the Delta pH pathway passengers cannot efficiently access the cpSec pathway.

PAGE 19

13 Studies have shown that the Delta pH pathway may be capable of transporting tightly folded precursors (Clark et al. 1997, Hynds et al. 1998). The natural precursor OE23 of the Delta pH pathway appears to be in a tightly folded conformation prior to transport (Creighton et al. 1995). Transport on the cpSec pathway appears require an unfolded precursor conformation (Ento et al. 1994, Hynds et al. 1998). So, it is also possible that Delta pH pathway precursor passengers carmot access the cpSec pathway because they have a tightly folded conformation. Protein Transport on the cpSec Pathway The belief that chloroplasts were origmated from a prokaryotic endosymbiont has driven speculation that thylakoid transport would be homologous to protein export from contemporary bacteria. The Sec system for protein export in E. coli has been investigated in detail by both biochemical and genetic techniques (Pugsley 1993, Rapoport et al. 1996). A preprotein translocase has been identified in E. coli (Brundage et al. 1990). The translocase consists of an integral membrane protein complex SecY/E/G and a peripheral membrane protein Sec A. A working model has been proposed by Hartl et al. (1990). In this model, chaperone SecB, by binding to the precursor m the cytosol, prevents the precursor from aggregation. SecA binds the precursor-SecB complex and brings the complex to SecY/E/G. ATP binds to SecA and its hydrolysis by SecA facilitates translocation of the precursor. Besides ATP, a proton-motive force is stimulatory for the Sec system operation. Some evidence suggests that SecA may function as a primary receptor for the precursor in bacterial protein transport. The direct interaction of SecA with the secretory

PAGE 20

* proteins was demonstrated by means of chemical cross-linking with EDAC (l-Ethyl-3[3-dimethlaminopropyl]-carbodiimide hydrochloride) and the interaction was signal peptide-dependent (Akita et al. 1990). A group of mutations in the Sec A gene (termed prlD mutants) suppressed certain precursor malE signal peptide mutations. This suggested that SecA directly interact with the precursor signal sequence (Fikes and Bassford 1989; Puziss et al. 1989). SecA exists in both cytoplasm and membrane. It was suggested that cytosolic SecA does not function as the precursor receptor since, in reconstitution studies, an excess of SecA did not compete with SecY/E-bound SecA for limited amounts of added precursor-SecB complex (Hartl et al. 1990). Thus, it has been inferred that membrane association activates SecA for its receptor function. SecA is also known as the translocation ATPase of protein transport, so SecA could work as both an ATPase and a receptor. There is also evidence to suggest that SecY is a precursor receptor. Antibodies against the N terminus of SecY are reported to prevent precursor binding to inverted membrane vesicles (Watanabe and Blobel 1989), as does trypsin treatment, which may inactivate SecY (Swidersky et al. 1990, 1992). Also, a group of mutations in the SecY gene (mutants prlA termed) suppress signal peptide mutations. By mapping the location of prlA mutations in SecY, Osborne and Silhavy (1993) hypothesized that the seventh transmembrane domain interacts with the hydrophobic core of the precursor signal peptide and plays a proofreading role. It is interesting that mutations in the SecE gene (prlG mutants) can also suppress signal peptides' mutations. So, it is possible that SecY or SecE may fiinction as precursor receptors.

PAGE 21

15 Thylakoid translocation of OE33 and PC has energy requirements similar to those of bacterial proteins that use the Sec system, implying that a Sec-Uke system is operational within chloroplast (Hulford et al. 1994, Yuan and Cline 1994a). Azide is known for its specific inhibition on SecA function in E. coli (Oliver et al. 1990). OE33 and PC transport is also inhibited by azide treatment, implying that a chloroplast homologue of SecA mvolved in the transport (Henry et al. 1994, Knott and Robinson 1994, Yuan and Cline 1994a). SecAand SecF-homologous genes were found in the chloroplast genomes of several algae (Robinson and Klosgen 1994) and subsequently the homologous genes were identified in plants (Berghofer et al. 1995, Nohara et al 1995, Laidler et al. 1995). The homologous genes in plants are nuclear encoded and named cpSecA and cpSecY. The requirement of cpSecA protein for OE33 and PC transport was clearly demonstrated in vitro with purified cpSecA from pea chloroplasts (Yuan et al 1994). Purified cpSecA replaced stromal extract and reconstituted transport of OE33 and plastocyanm with bufferor ureawashed thylakoids m an azide-sensitive manner. Haward et al. (1997) observed a cpSecA-precursor crosslinking product during precursor binding. This at least indicates that the precursor is in direct contact with cpSecA. In vivo evidence for the cpSecA's role on the cpSec pathway protein transport came from the research with a maize mutant thai: the maize thai mutant was selectively inhibited in transport of OE33, plastocyanm and PSI-F (Voelker and Barkan 1995); the Thai gene was isolated and shown to encode maize cpSecA (Voelker et al. 1997).

PAGE 22

16 Protein Transport on the Delta pH Pathway The Delta pH pathway was first revealed by the discovery that two lumen proteins, 0E17 and OE23, were transported into the thylakoids in such a unique manner that no soluble factor or NTPs were required for the transport. The ApH across the thylakoid membrane was the sole energy requirement (Cline et al. 1992). Little is known about the operating mechanism for the Deha pH pathway. It appears to work like a typical export system in E. coli or ER. First, precursors using the system have classical signal peptides (Cline and Henry 1996); second, the precursor transport uses loop topology and is likely translocated into the lumen from amino terminus to the carboxyl terminus (Fincher et al. 1998, Fmcher and Cline unpublished). Although the Delta pH pathway does not require any soluble components, it certainly requires proteinaceous components on the thylakoid membrane. Transport cannot be conducted with protease pre-treated thylakoids (Robinson et al. 1996). Also, transport is saturated with high level of precursor and competed by unlabeled precursor (Cline et al. 1993). A component named Hcfl06 was identified as essential for Delta pH pathway function. Mutation in the Hcfl06 gene results in high-chlorophyll fluorescent (hcj) phenotype and causes accumulation of Delta pH pathway intermediate precursors in the stroma (Voelker and Barkan 1995). Although the mutation diminishes the ability of the thylakoids to generate and maintain a transmembrane pH gradient, the ApH present was as high as in Thai, which transports precursor at normal rate on the Delta pH pathway. Isolated chloroplasts from hcfl06 mutant seedlings were totally unable to transport 0E17 on the Delta pH pathway, but were still capable of transporting OE33 on the cpSec pathway (Settles et al. 1997). The Hcfl06 gene was cloned and the antibodies against Hcfl06 localized the protein on

PAGE 23

17 thylakoid membranes. The bulk of the Hcfl06 protein is exposed to the stroma, suggesting that it may function as a receptor. Precursors on the Delta pH pathway have a critical RR motif in their lumen-targeting domain. If Hcfl06 functions as a receptor, it is likely that the RR is interacting with Hcfl06 during the targeting process. The Delta pH pathway was once considered to be eukaryotic innovation having evolved after endosymbiosis because all of its known substrates are absent from cyanobacteria, from which the chloroplast is believed to be evolved. However, database searches with the maize Hcfl06 sequence revealed a homologous class of hypothetical bacterial proteins. This evidence strongly suggested that a similar pathway is operating in bacteria. The existence of the system in bacteria was also predicted by Berks (1996) based on the observation that a class of periplasmic proteins in bacteria are synthesized with signal peptides containing an RR motif These redox proteins are apparently exported together with their redox cofactors and the exported proteins may be in a folded conformation. E. coli contains three homologues of Hcfl06 TatA, TatB and TatE, where tat stands for twin-arginine translocation. tatA and tatB genes are located in a four-gene operon, whereas the tatE is unlmked. tatA and tatE gene products have recently been shown by Sargent et al. (1998) to be required for the export of a range of proteins bearing the twin-arginine motif in their signal peptides. The Sec-system was shown to be unaffected in the mutant strains in which these genes were disrupted. Mutation of tatB also resulted in defective transport of RR-precursor (Weiner et al. 1998). Disruption of the tatC gene, which is also a member of the tat operon, results in a complete block in transport of five tested RR-precursor (Bogsch et al. 1998). TatC is a multispanning membrane protein and its homologous genes are present in a range of bacteria, plastids,

PAGE 24

IS and mitochondria. Thus the Delta pH pathway is a novel pathway, discovered in plant thylakoids, but apparently exists in prokaryotes. Summary and Perspective Protein transport is required for the biogenesis of the chloroplast, the site of photosynthesis. Hundreds of chloroplast proteins are encoded in the cell nucleus and are synthesized in the cytosol. These proteins have to be transported into the chloroplast and functionally assembled. A nuclear-encoded chloroplast protein is initially synthesized as a precursor protein that contains a transient transit peptide at its amino terminus. For a thylakoid lumen protein, its transit peptide consists of two parts: one is a stroma-targeting domain (STD) and another is a lumen-targeting domain (LTD). Import of the precursor across the chloroplast envelope is governed by the STD of the transit peptide and accomplished by the cooperation of machinery components on both outer and inner envelope membranes. Such machinery is collectively called the general import apparatus. ATP hydrolysis is used to power import. The STD is cleaved by a processing protease in the stroma, resulting in either a mature stromal protein or an intermediate thylakoid protein precursor which is fiirther directed to the thylakoids by the exposed LTD. There appears to be one major pathway for protein import across the chloroplast envelope. However, there are two pathways for protein transport into the thylakoid lumen. One pathway is called the cpSec pathway since this pathway employs a chloroplast homologue of the bacterial SecA protein. A second pathway is named the

PAGE 25

19 Delta pH pathway because in this pathway, the ApH across the thylakoid membrane is the only energy requirement for protein transport. Each pathway is specific for a subset of precursor proteins. Protein transport into the thylakoid lumen is directed by the LTD. All precursors on the cpSec pathway or Delta pH pathway have a similar basic LTD structure containing three essential domains: N, H and C. Targeting to the cpSec pathway requires a specific hydrophobic H domain. Targeting to the Delta pH pathway requires a specific N domain (Henry et al 1997). A critical RR motif in the N domain is required for committing the precursor to the Delta pH pathway (Chaddock et al. 1995). The RR motif is thought to interact with a putative receptor on the thylakoid membrane. Further information is necessary to establish how the LTD is interacting with transport machinery and whether the precursor mature part is involved in the targeting step. The cpSec pathway is powered by ATP hydrolysis and stimulated by ApH (Cline et al. 1992). The soluble component cpSecA is required for the transport (Yuan et al. 1994). Chloroplast homologues of bacterial SecY have been cloned (Laidler et al. 1995) and recent studies show that cpSecY is functional on the cpSec pathway (Hiroki et al. in preparation). Although Haward et al. (1997) demonstrated that precursor can form a complex with cpSecA on the thylakoid membrane, it was not clear from that study whether the cpSecA functions as a receptor or even if the interaction between precursor and cpSecA is specific. Also it is not known whether the mteraction involves cpSecY. Further work is required to show how cpSecA engages precursors in targeting and translocation.

PAGE 26

20 The ApH is the only energy requirement for the Delta pH pathway transport and no soluble component is required (Cline et al. 1992). The thylakoid protein Hcfl06 was recently identified as a component required for the pathway (Settles et al. 1997). Although the Delta pathway was first discovered by a biochemical approach, the machinery components were identified by genetic methods. However, development of new biochemical approaches is necessary to delineate the role of identified components in the translocation mechanism. In the studies presented in this dissertation, I have conceptually and experimentally divided protein transport across the thylakoid membrane into two steps. The first is targeting in which the precursor is committed to the pathway. The second is translocation, wherein the protein actually is moved by the transport machinery across the membrane. I hypothesize that targeting results fi-om a specific interaction between the precursor and components of the protein transport machinery. Each protein transport pathway has its own components to recognize the precursor and such components are referred as precursor receptors. So far, protein transport machinery components (receptors) related to pathway selection have not been identified. The purpose of this study is to examine the molecular determinants and steps of the process that result in precursor commitment on the cpSec pathway or the Delta pH pathway. The specific questions addressed are: 1 . Is there an earhest detectable precursor targeting step that commits the precursor on the cpSec pathway or the Delta pH pathway? Specifically, is there a detectable complex formed during the precursor targeting step? 2. Are there any receptors that interact specifically with the precursor during the precursor

PAGE 27

21 targeting step? 3. What are the elements of precursors necessary for targeting? 4. What are the energy requirements for precursor targeting and translocation? The long range goal is to determine underlying reasons for the existence of multiple pathways for protein transport into the thylakoids. To achieve the goal, it will first be necessary to define each pathway. The successfiil completion of this study will provide fiirther understanding of thylakoid protein transport mechanisms.

PAGE 28

CHAPTER 2 PROTEIN TARGETING AND TRANSLOCATION ON THE cpSEC PATHWAY Abstract To investigate the basis for pathway-specific targeting, the characteristics of membrane binding and chase of precursors on the cpSec pathway were examined. Previous studies showed that the cpSec pathway precursor iOE33 could form a productive intermediate and be crosslinked to cpSecA on the thylakoid membrane (Haward et al. 1997). Our results support the notion that precursor commitment to the thylakoid cpSec pathway occurs on the membrane, rather than in the soluble stroma. Precursor binding to the thylakoid membrane required stromal protein, of which cpSecA was the essential component, and occurred when transport was inhibited by removing ATP from the assay with apyrase. Sodium azide and ionophores, although inhibiting transport, did not result in increased precursor binding. Subsequent transport of bound precursor required ATP, but was not stimulated by additional cpSecA. Upon interaction with thylakokls, precursors could be crosslinked into a high molecular weight complex (cpSec complex) that migrated anomolously in SDS-PAGE. The crosslinked product was immimoprecipitated with antibodies to cpSecA as well as with antibodies to cpSecY. Formation of the cpSec complex was strictly precursor-specific. Precursors capable of being transported by the cpSec pathway were crosslinked into the complex, whereas precursors transported by the Delta pH pathway and SRP pathway were not. Of interest is the fact that precursors consisting of a cpSec-compatible signal peptide fused to a Delta 22

PAGE 29

23 pH passenger protein produced a higher molecular weight crossUnking product that was not immunoprecq)itated by antibodies to cpSecA. Previous studies have shown that Deka pH passenger proteins are incapable of efficient transport on the cpSec pathway. This later result suggests that commitment to the cpSec pathway at an early step involves not only the signal peptide, but elements of the passenger protein as well. Introduction Nucleus-encoded thylakoid lumen-resident proteins are synthesized in the cytosol as precursors with bipartite amino-terminal transit peptides. Precursors are localized to the lumen by a two-step mechanism (Cline and Henry 1996, Schnell 1998, Robinson et al. 1998). In the first step, the precursors are imported across the chloroplast envelope into the stroma. This step is governed by the stromal targetii^ domain of the transit peptide. A stromal processing protease removes the stromal targeting domain, thereby producing a stromal intermediate precursor, which is transported across the thylakoid membrane in the second step. Thylakoid transport is directed by the lumen-targeting domain of the transit peptide, which is removed by the thylakoid processing protease. The cpSec pathway is one of two pathways for protein transport into thylakoid lumen. OE33, PC and PSIF (F sub-unit of Photosystem I) are three proteins known to use the cpSec pathway for their transport. Protein transport on the cpSec pathway requires ATP, a stromal protein cpSecA (Yuan et al. 1994), and a membrane protein cpSecY (Mori et al., in preparation). According to Haward et al. (1997), when ATP was eliminated with apyrase, iOE33 transport was inhibited, resulting in accumulation of boxmd precursor on the thylakoid membrane. The bound precursor could be chased into

PAGE 30

24 thylakoid lumen upon ATP addition. Precursor binding is considered to be a targeting step whereas the chase is the translocation step. cpSecA and cpSecY are homologues of the bacterial Sec A and SecY proteins (Berghofer et al. 1995, Nohara et al. 1995, Laidler et al. 1995). The bacterial Sec A protein is a translocation ATPase that binds to precursors and SecY and "pushes" peptide segments across the membrane through a cycle of insertion and deinsertion. SecY is a membrane protein that is thought to play a major role in forming the protein translocation channel with SecE component (Akiyama and Ito 1985, Meyer etal. 1999). Committing a precursor protein to a specific pathway, i.e. targeting, is believed to be initiated by an interaction between precursor and a receptor. It has been shown that the lumen-targeting domain determines whether a protein is transported on the cpSec or Delta pH pathway (Henry et al. 1994). It has also been shown that the mature protein domain, i.e. the passenger, can greatly affect the efficiency of transport by the cpSec pathway. For example, although a cpSec LTD directed transport of 0E17 on the cpSec pathway, the transport was inefficient. Secondly, the hybrid (DT) transit peptide, while directing efficient transport of Sec passengers on both pathways, was only able to direct efficient transport of Delta pH passengers on the Delta pH pathway (Henry et al. 1994, 1997). Haward et aL (1997) reported that iOE33 formed a complex on the thylakoid membrane with cpSec A and suggested that cpSecA may fimction as a receptor. However, it was not known which part of the precursor boimd to cpSecA and more information is required to determine whether cpSecA functions as a receptor. I am interested in learning how the cpSec pathway system recognizes its respective substrates and commits them to the pathway. I have attempted to describe and

PAGE 31

25 characterize the first committed step on the cpSec pathway and to define in general terms the sequences reqiiired for commitment. My results show that the first observable interaction of precursors with the cpSec pathway occurs on the membrane and results in formation of a large complex that includes precursor, cpSecA, and cpSecY. This interaction requires a cpSec LTD and additionally a cpSec-compatible flanking region. Proteins that possess dual targeting signal peptides fused to Delta pH passenger proteins are capable of binding to the membranes as well as chasing into the limien on the delta pH pathway. However, these proteins do not form a complex with cpSecA. Materials and Methods Preparation of precursors In vitro transcription plasmids for iOE33, iOE23 and LHCP were described as Cline et aL (1993). Preparation of tPC, DT33, DT17 and DT23 were described as Henry et al. (1997). Preparation of Chloroplasts, Lysates, Thylakoids and Stroma Chloroplasts were isolated fi-om 9 to 10-day-old seedlings of pea (Laxton's Progress 9). Lysates, thylakoids and stromal extract were prepared from the intact chloroplasts (Chne et al. 1993) Preparation of Purified cpSecA CpSecA was purified from stromal extract as described by Yuan et al. (1994b), except that studies reported here used cpSecA obtained after the Mono-Q ion exchange step. The concentration of purified cpSecA was estimated by Coomassie staining of SDS polyacrylamide gels using BSA as a standard.

PAGE 32

26 Thylakoid Protein Transport Assay Transport assays were conducted essentially as described previously (Cline et aL 1993): 25 ng chlorophyll of thylakoids or lysates in 25 ^il of import buffer (50 mM Hepes buffer pH 8, 0.33 M sorbitol) was mixed with 30 ^l HKM buffer (10 mM Hepes buffer pH 8, 10 mM MgCk) with or without 300 fig of stromal protein, and incubated with 20 ^^l 1 fold diluted in vitro translated precursor in import buffer with 30 mM leucine. ATP or ^yrase, ionophores, or azide were added in respective assays. The total volume of ') 1 each assay was about 80 yA. Assays were incubated under 70 m' s' white Ught (or dark) for 15-30 minutes at 25°C. After incubation, thylakoids were recovered by centrifiigation and were washed once with import buffer. One half of each sample of recovered membranes was analyzed directly on SDS-PAGE. The other half was treated with 10 ^g thermolysin in 100 (xl of import buffer (Cline 1986) and then analyzed by SDS-PAGE and fluorography. ' Precursor Binding and Chase Assays Thylakoids or chloroplast lysate (in 25 \i\ import buffer) equivalent to 25 ^g chlorophyll was mixed with 40 ^1 HKM buffer with or without 400 jxg of stromal protein. Two |ag cpSecA, ionophores, or azide were added to respective assays. The mixture was incubated for 10 minutes in the dark in the presence of 2 |il (units) apyrase. About 10 \il in vitro translated precursor was added and the incubation continued for 15 minute at 25°C in the dark. Following the incubation, thylakoid membranes were recovered by centrifiigation, washed twice with import buffer, and divided into two equal portions in fresh microfuge tubes. One portion was analyzed directly. The other portion was incubated under chase conditions. The chase was conducted under 70 ^E m"^s"' white

PAGE 33

light for 15-30 minutes at 25°C in a total of 37.5 jil of import buffer containing -200 ^ig protein of stromal extract and ImM DTT. Where designated, 12.5 ^g chlorophyll aliquots of thylakoids recovered from binding or chase samples were treated with 10 ^ig thermolysm in 100 fil of import buffer (Cline 1986). Preparation of Urea-washed or Thermolysin Treated Thylakoids for Binding Assays and Urea or Na2C03 Wash of the Thylai^oid Bound Precursor Urea-washed thylakoids were prepared by incubatmg 0.5 mg chlorophyll of thylakoids in 1 ml of 2 M urea in import buffer for 8-10 minutes on ice. Thylakoids were recovered by centrifiigation and washed once with import buffer. Thermolysin-treated thylakoids were prepared by incubating 0.33 mg chlorophyll of thylakoids in 1 ml import buffer containing 100 ng thermolysin on ice for 40 minutes. Thylakoids were recovered by centrifiigation and washed twice with import buffer containing 10 mM EDTA and once with import buffer. Urea or Na2C03 wash of the thylakoids recovered from binding assays was accomplished by incubating 0.5 mg chlorophyll/ml of thylakoids with 6 M urea or 0. 1 M Na2C03 (in water) on ice for 30 minutes. The thylakoids were recovered by centrifiigation (at 3200x G for 8 minutes) and washed twice with import buffer. Cross-linking Reactions Binding assay mixtures, following incubation for 15 minute, received 0-2 mM crosslinker from 0-40mM stocks in DMSO (for DSS (disuccinimidyl suberate), MBS (mMaleimidobenzoyl-N-hydroxysuccinimide ester) and APDP ( N-[4-(pAzidosalicylamido) butyl]-3'-[2'-pyridyldithio] propionamide) ) or H2O (for BS^ (bis [sulfosuccinimidyl] suberate)). Cross-linking was allowed to proceed at room temperature for 30 min or at 0"C for 2 hours and was termmated with 50 mM Tris-HCl

PAGE 34

28 pH 7.5 from a 1 M stock. After the cross-linking reaction, the memlM-ane fraction was recovered by centrifiigation and washed with import buffer. Preparation of Antibodies LHCP and CpSecA antibodies were prepared as described by Payan and Cline (1991) and Yuan et al. (1994b), respectively. Peptides NH2CKLQDLQKKEGEAGRKK-COOH and NH2-CDDVSEQLKRQGASIPLVRPGKCOOH, termed Inl and In2 respectively, correspond to two internal stromal-facing regions of cpSecY. Inl, In2 and the antibodies against the peptides were prepared by BioSynthesis company (TX). Inl and In2 were conjugated via their N-terminals to carrier keyhole limpet hemocyanin with crosslinker MBS and the crosslinked products were used as antigens. The peptide NHz-CRAEnSQKYNIELYDFDKY-COOH (termed Cterm), corresponding to the C-terminal stromal-facing region of pea cpSecY, was made and cross-linked by Genosys Biotechnologies company (Woodlads, TX). The antibodies raised against the Keyhole limpet hemocyaninlinked C-term peptide were prepared by Cocolico Company and described by Mori et al. (In preparation). Immunoprecipitation of the Cross-linked Complex Twenty-five \ig chlorophyll of thylakoid membrane recovered from each crosslinking assay was resuspended in 75 ^1 of 10 mM HEPES/KOH (pH 8), 10 mM MgCk, followed by 4 ^1 20% SDS. After 30 minute incubation at 3TC, the sample was brought to 800 ^l with 10 mM HEPES/KOH (pH 8), 10 mM MgCh, 0.2% Triton X-100. Ten to fifteen ^1 serum, with or without 10-20 [ig synthetic peptides, was added and the sample was mixed end-over-end at 4"*C overnight. Forty fjl 1:1 protein A Sepharose slurry in 10 mM HEPES/KOH (pH 8), 10 mM MgCla was added and the sample was mixed end-

PAGE 35

29 over-end at 4°C for 1 hour. The Sepharose beads were pelleted by centrifugation at 500x G for 3 minutes, resuspended in 10 mM HEPES/KOH (pH 8), 10 mM MgCl2, transferred to a new tube, pelleted, and then treated with 20 ^1 2X SDS sample bufifer. Anatysis of Samples Samples recovered from the assays were subjected to SDS-PAGE and fluorography. Most san::^les were analyzed with 12.5% acrylamide concentration gels. Cross-linking and immimoprecipitation samples were analyzed with 7.5% gels. Quantification of the amount of radiolabeled proteins resulting from these assays was accomplished by scintillation counting of radiolabeled proteins extracted from excised gel bands (Cline 1986). Results No Interaction can be Detected between cpSec Pathway Precursor and Soluble cpSecA CpSecA is the only soluble chloroplast component required for protein transport on the cpSec pathway and, therefore, the only candidate for a specific interaction in the soluble fraction. Bacterial SecA is reported to interact weakly with precursor in solution (Hartl et aL 1990) and can be crosslinked to precursors with ED AC (l-Ethyl-3-[3dimethlaminopropyl]-carbodiimide hydrochloride) (Akita et aL 1990). In preliminary studies, we were unable to detect any interactions between cpSec pathway precursors and cpSecA in solution using several different interaction assays. These included nondenaturing PAGE (gel shift assays), gel filtration of the authentic stromal intermediate

PAGE 36

30 isolated from chloroplasts, crosslinking combined with immimoprecipitation, and nascent chain crosslinking combined with immunoprecipitation (data not shown). iOE33 Binds to the Thylakoid Membrane when Transport is Prevented by Eliminating the ATP In E. coli, strong binding to Sec components occurs on the membrane (Hartl et aL 1990). In addition, Haward et al. (1997) reported that the precursor iOE33 binds productively to thylakoids and can be crosslinked to cpSecA. Here we have fiuther characterized this binding and investigated the possibility that membrane binding represents the first committed step on the cpSec pathway. [^H]-labeled precursor was incubated with thylakoids under assay conditions either conducive for transport or where a single transport requirement was withheld (Figure 2-1). Wheat iOE33 was used as the precursor as it is the most efficient cpSec pathway substrate imder our conditions. As in previous studies (Yuan et al. 1994a, Hulford et al. 1994), iOE33 was transported into the thylakoid lumen when stromal extract, ATP and ApH were present, as judged by processing to the mature size and inaccessibility to exogenous protease. Transport was inhibited when assays were conducted in the presence of ionophores that dissipate ApH, or azide, a SecA inhibitor, or m the absence of ATP (apyrase was used to scavenge all traces of ATP). In the absence of ATP/presence of apyrase, precursor binding to the thylakoids membrane was stimulated (Figure 2-1, lane 1 and lane 4). The amoimt of boimd precursor was ~ 2.6 fold higher in the presence of apyrase than that under any of the other conditions. In the presence of apyrase, about 2.6% of the added precursor in the transport mixture was boimd on the thylakoid membrane. Of interest is that, although

PAGE 37

31 light + + + + + ATP + + + + nig/val •+ SE + + + + + apyrase + + azide _ _ _ _ _ + /iOE33 \mOE33 — mOE33 Figure 2-1. qsSec-pathway transport intermediate accumulated on the thylakoid membrane when transport was arrested with apyrase. Transport assays with [^H]-leucine labeled iOE33 precursor were conducted with isolated thylakoids under conditions (shown above the panel) designed to test the effects of removii^ ATP or stromal extract (SE), of dissipating the ApH, or of inhibiting cpSecA with azide. Assays (75(il) contained 412.5 |ig protein of stromal extract (SE), 5 mM Mg-ATP, nigericin and valinomycin (nig/val) at 0.5 |j,M and 1 ^M, respectively, and 10 mM sodium azide as indicated above the panel Apyrase treatment was 2 unit per 75 ^1 assay. For the top panel, the thylakoid membranes were recovered by centrifiigation and washed with import bxiflfer. Membranes in the lower panel were treated with the protease thermolysin prior to washing. Sanq>les were then analyzed with SDS-PAGE and fluorography. Each lane contained 12.5 jig chlorophyll (100%) of thylakoid membranes present in each assay. Lane P represents 0.25 ^l (5%) precursor added to the assay reaction. The positions of the precursor iOE33 and the mature form (mOE33) are marked.

PAGE 38

32 ionophores and azide inhibited transport of iOE33, they did not result in increased binding. Binding of iOE33 to Thylakoids is Productive and Stimulated by cpSecA To determine if precursor was productively bound in the absence of ATP/presence of apyrase, membranes, washed following a binding assay, were incubated with stromal extract and ATP at 25°C for 30 minutes (Figure 2-2). Approximately 1 1% of the bound precursor was subsequently transported (chased) into the lumen and processed to mature size, whereas precursor boimd without stromal extract or cpSecA failed to be chased into the lumen (Figure 2-2/4). cpSecA is the required stromal con^nent for iOE33 transport (Yuan et aL 1994b). Purified cpSecA significantly mcreased precursor binding and the subsequent chase over that of buffer alone. The amount of cpSecA added is approximately equivalent to the amount in imfractionated stromal extract. The relatively low percentage of precursor that is chased under our conditions may reflect the feet that transport competence of thylakoids is significantly reduced by the washing steps. This is shown in Figure 2-25. A transport assay conducted with apyrase treated and bufferwashed thylakoids plus stromal extract yielded ~56% transport compared to freshly prepared membranes (con^are lane 1 to lane 4). It was shown in Figure 2-1 that azide or ionophores alone, while inhibiting transport, did not result in increased precursor binding. Figure 2-3 examines the effects of azide and the ApH on binding in the absence of ATP/presence of apyrase. Azide slightly decreased, whereas ionophores had no significant effect on binding. Precursor bound under all those conditions was chased into the lumen, although with varying efficiencies. Of interest is

PAGE 39

33 Figure 2-2. cpSecA stimulates iOE33 binding on the thylakoid membrane and the binding is productive. Panel A: 15 ^1 [^H]-labeled iOE33 precursor in vitro translation product was incubated with 37.5 ^g chlorophyll of thylakoids (in 37.5 ^1 import buffer) in the presence of 3 ^il (equal to 3 units) apyrase in import buffer for 15 minute at 25*'C in a total 1 15.5 ^il binding assay. Binding assays received -800 ng of stromal protein in 60 ^1 10 mM Hepes/KOH pH 8, 10 mM MgCb (HKM buffer) (lanes 1, 4, 7), or 60 \i\ HKM alone {lanes 2, 5, 8), or ~3fig purified cpSecA in 60 \il HKM buffer (lanes 3, 6, 9). After binding incubation, the thylakoid membranes were recovered, washed twice with import buffer, divided into three equal aliquots and transferred to new tubes. One aUquot was directly analyzed (lanes 1-3). A second aliquot was treated with thermolysin (lanes 4-6). The third aliquot was incubated under chase conditions and then treated with thermolysin (lane 7-9). Each lane contains 100% of thylakoid membrane from each assay. Lane p represents about 0.25 precursor in vitro translation product. Panel B: Thylakoid transport assays conducted with fresh thylakoids (Lanes 1, 2, 3) or thylakoids incubated with apyrase under conditions of a binding assay and then washed twice with import buffer before use (lane 4). All reactions contained 5 mM ATP. Reactions of lanes 1, 4 contain stromal extract; lane 2 buffer; lane 3 purified cpSecA. Final samples were treated with thermolysin before being analyzed by SDS-PAGE and fluorography. The charts display radioactivity (CPM) of corresponding bands.

PAGE 40

34 Binding Binding Chase 1234 1234 1 234 Protease I + + ++ + + + + post-treatment Figure 2-3. The effects of ApH and azide to iOE33 binding to the thylakoid membrane. iOE33 binding assays were conducted in the presence of stroma and apyrase. Binding assays in lane 2, 3 and 4 were supplemented with 3 CCCP (Carbonylcyanide 3chlorophenylhydrazone), 0.5 (xM/l^iM nig/val or 10 mM sodium azide respectively. Chase assays were all conducted under identical condition (see Methods).

PAGE 41

35 that precursor bound in the presence of ionophores was chased into the lumen with efficiency greater than precursor bound under other conditions. Because ionophore effects of nig/val persist through washing steps, this was the first indication that a ApH was not required for the chase reactioiL Soluble cpSecA is not Required for the Chase of Bound Precursor The requirements for chase of boimd precursor into the thylakoid liunen were investigated (Figure 2-4). Maximum chase of bound iOE33 resulted when stromal extract was included in the re-incubation mixture. ATP alone was able to partially stimulate chase, but chase with ATP plus stromal extract was no greater than stromal extract alone. Purified cpSecA was ineffective in stimulating chase, indicating that cpSecA is committed at the early binding step and that free cpSecA is largely ineffective at the chase stage. This is consistent with observations in the bacterial Sec system that excess soluble SecA did not compete with membrane-bound SecA for limited amounts of added precursor-SecB complex (Hartl et al. 1990) and that bacterial SecA does not cycle off the membrane during protein transport (Chen et al. 1996). Although free cpSecA was not required for the chase, bound cpSecA played an important role during the chase since azide partially inhibited the chase (Figure 2-4B). Unexpectedly, the chase was stimulated by ionophores. Yet, the thylakoids' ability to generate a ApH was not likely destroyed by manipulations involved in binding and washing because apyrase treated and washed thylakoids were still capable of transporting fresh precursor in an ionophore-sensitive fashion (data not shown). Rather, the energy required for transport was derived from ATP because addition of apyrase prevented chase of precursor into the lumen (data not

PAGE 42

36 Figure 2-4. Requirements for chase of bound precursor into the lumen. A. 28 \il [^H]leucine labeled iOE33 precursor was incubated with 87.5 ng chlorophyll of chloroplast lysate (in 1 10 fil import buffer) and -1200 jjg protein of stromal extract (in 120 HKM buffer) in the presence of 7 units (nl) apyrase for 15 minute at 25°C. Thylakoid membranes were then recovered and washed twice with import buffer and divided into seven equal portions. Each portion was transferred to a new tube. One portion was analyzed directly (lane 7). The remaining portions were further assayed for chase under different conditions. Assays contained import buffer (lanes 2, 4), 200 ^g stromal protein (SE)(lanes 3, 5), -0.8 \xg purified cpSecA (lanes 6, 7) were incubated in the presence {lanes 4, 5, 7) or absence (lanes 2, 5, 6) of 5 mM ATP for 30 min. at 25°C in light in a total 37.5 |al. Recovered thylakoids were treated with the thermolysin and then analyzed by SDS-PAGE and fluorography. B. The binding incubation was similar to that shown in panel A. During the chase step, 12.5 ^g chlorophyll of thylakoid membrane with bound precursor were resuspend in buffer containing 200^g protein of stroma extract and 5 mM ATP plus 1 (iM/0.5 |xM nigericin/valinomycin (t^N) or 10 mM sodium azide (Azide) or import buffer (IB).

PAGE 43

37 shown). Stromal extract contains small amounts of ATP and presumably this was sufficient to power the transport reaction in assays containing stromal extract alone. Precursor Binding to Thylakoids is Mediated by Protein-protein Interactions Further investigation into the nature of the observed precursor binding is shown in Figure 2-5. Binding was maxunal with membranes that were either bufferor ureawashed and when stromal extract was present in the reaction mixture. Approximately 2.12.7 % of the added precursor was bound to the membrane under these conditions. Binding to protease-treated membranes was greatly reduced (only -0.4% of the added precursor was boimd to the membrane). Following the binding reaction, thylakoids were washed with 0.1 M NaiCOs or 6 M urea to assess the nature and strength of the binding. The majority of precursor bound to bufferor ureaprewashed membranes (~60%) was removed by the NaaCOs and urea washes, whereas the low level of precursor bound to protease-treated membranes was largely resistant. This, combined with previous observations that transport is eliminated by protease (Robinson et al. 1996), but not ureatreatment of thylakoids (unpublished data) suggests that iOE33 binding involves proteinprotein interaction, whereas a low level of precursor can non-specifically interact with the thylakoid surface. Productive Binding Results in Formation of a Complex on Thylakoids The above results imply that binding of iOE33 involves cpSecA and thylakoid protein(s), and suggests the formation of a complex on the membrane, similar to the Sec complex shown for bacterial membranes (Brundage et aL 1990). To assess whether such a complex exists, a binding reaction was conducted for 15 minutes, after which the cross-

PAGE 44

38 Treatment s to the thylakoids before the binding IB wssh Urea wash Protease incubation (lane 1-6) (lane 7-12) (lane 13-18) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 SE+ + + + . + + + Bound iOE33 US ^ IB + Thylaltoids wash after the binding ^^^l^^a Urea + + + + + + + + + + + + + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Figure 2-5. iOE33 precursor binding to the thylakoid membrane is mediated by proteinprotein interactions. Nine ^1 [^H] labeled iOE33 translation product was incubated with 37.5 (ig chlorophyll of import buflferwashed thylakoids or 2 M ureawashed thylakoids or thermolysin (100 ^ig/ml)treated thylakoids in the presence or absence of -525 ^g stromal proteins in a total 1 12.5 ^il. All assays contained 3 unit of apyrase and were incubated in the dark at room tenq)erature for -30 min. After the incubation, each sanq)le was divided into three equal portions. The thylakoid memtMranes in each portion were washed with import buffer {lanes 1, 4, 7, 10, 13, 16) or 0.1 M sodium carbonate {lanes 2, 5, 8, 11, 14, 17) ox 6 M urea {lanes 3, 6, 9, 12, 15, 18). Thylakoid membranes recovered from the assays (12.5 ^ig chlorophyll) by centrifiigation were analyzed by SDS-PAGE and fluorography.

PAGE 45

39 linking agent DSS (disuccinimidyl suberate) was added to stabilize any possible con^lex. Multiple cross-linking products, apparent as higher molecular weight radioactive bands, were present in both the sohible, stromal fraction and the thylakoid membrane fraction (Figure 2-6 A, (1) and (2)). Because of the requirement for cpSecA in the binding reaction, crosshnked products were subjected to immimoprecipitation with antibodies to cpSecA. Soluble crossUnking products were not immimoprecipitated (Figure 2-65). However, the large crossUnking product that migrates near the top of the separating gel was immunoprecipitated from the membrane fraction with antibodies to cpSecA (Fi^e 2-6^4, (3)) but not with preimmune serum (data not shown, but see Figure 2-7). Further experunents (Figure 2-65) indicated that the formation of this crosslinked product required both stromal extract and the thylakoid membrane and that purified cpSecA could replace stromal extract in this reaction. From these data, it can be concluded that iOE33 forms a complex with other proteins on the thylakoid membrane and that cpSecA is part of the complex. In a standard binding assay containing thylakoids and stromal extract, -2.4% of the added precursor remained bound to the recovered and washed thylakoid membrane. When 1 mM DSS was added at the end of the binding reaction, -3.5% of the added precursor was recovered as the cross-linked complex. Over 50% of the cross-linked complex was immunoprecipitated with antibody against cpSecA. When thylakoids recovered from a binding assay were treated with DSS following the washing steps, the same cross-linked complex was formed, though a lesser amount than when crosslinker was added directly to the binding reaction (data not shown). When the binding assays were conducted in the presence of ionophores or sodium azide alone, cpSecA-containing

PAGE 46

40 (1) fl'? iWP 11 (2) (3) DSS(mM) 0 0.10.5 1 2 0 0.1 0.5 1 2 0 0.10.5 1 2 SE S M S M B Figure 2-6. Formation of a complex between precursor and con^wnents of the cpSec machinery. A. Crosslinkers stabilize a large cpSecA-contaming complex on the membrane. Ten |il [^H] labeled iOE33 in vitro translation product was incubated with 25 ^g chlorophyll of chloroplast lysate plus 40 ^.1 (400 ng of protein) additional stromal extract in the presence of 2 units (in l^l import buffer ) apyrase. After 15 minutes incubation, 4 \xl of varying amoimts of DSS in DMSO were added to the reaction mixture, which was further incubated for 2 hours in an ice bath. The final DSS cocentrations in the mixture were 0-2 mM. The cross-linking reaction was terminated by adding 4 )al 1 M Tris-HCl, pH 7.5. The membrane and stromal fractions were separated by centrifugation and aliquots of the samples were further analyzed by anti-cpSecA immunoprecipitation. Final samples were analyzed by SDS-PAGE (7.5% gels) and fluorography. Panels: (1), the cross-lmking products in 7.5 ^1 supernatant from crosslinking mixture (2), the cross-linking products on the 6.25 |ig chlorophyll of membrane; (3), the antiSecA immunoprecipitation products of the cross-Unking products on the about 12.5 |4,g chlorophyll of membrane. B. cpSecA is required for con^lex formation. Ten ^1 [^H] -labeled iOE33 precursor was incubated either with stromal extract (SE, -600 ^g protem ) or with 25 ^g chlorophyll of thylakoids (T) or with the thylakoids plus purified cpSecA (T+SecA, 1.6 ^g cpSecA) or with the thylakoids plus stroma extract (T+SE) and the reactions were crossUnked with 1 mM DSS. The reactions were then subjected to immunoprecipitation with antibody to cpSecA. Immunoprecipitation products from the soluble fraction (S) or the membrane (M) are shown.

PAGE 47

41 crosslinking products were not obtained (data not shown), indicating a strong correlation between productive binding and the ability to form a crosslinked con^lex. cpSecY is Part of the cpSec Complex In bacteria, the interaction of precursor and SecA with the membrane results in a complex with the SecY/E/G protein. To determine if the precursor-cpSecA complex also contains cpSecY, immunoprecipitations were conducted with antibodies generated to cpSecY. Antibodies were raised to synthetic peptides that correspond to several regions of the predicted pea cpSecY sequence (see Methods). Initially, antibodies were raised to peptides corresponding to two internal stromal-facmg regions of cpSecY. These antibodies are capable of specifically immunoprecipitating the cpSecY translation product but fail to give a band on immimoblots (Mori and Cline, impublished). Subsequently, a third antibody was raised to the extreme carboxyl terminus of cpSecY. This antibody immxinodecorates a 42 kD pea thylakoid protein, presumed the cpSecY, located in the stroma lamellae (Mori et al., in preparation). All three antibodies immunoprecipitated the large precursor cpSecA complex (Figure 2-7 A and B). The con^lex was not precipitated with pre-immune serum or when immunoprecipitation was conducted in the presence of the corresponding synthetic peptides. To verify that DSS cross-linker was not simply crosslinking the boimd precursor to any component on the thylakoid membrane, immunoprecipitations were conducted with antibodies against LHCP, a major protein of the thylakoid membrane but not related to the cpSec pathway. No radiolabeled cross-Unking products were unmunoprecipitated with these antibodies (Figure 2-7C).

PAGE 48

42 antiSccY Sent Prclnl Inl 1 2 Inl antiSecA Pre Im 6 7 I antiLHCP XSI XMI B cpSecY synthetic pq)tide XP antiSecY C-term antiSccA Im Im Pre Im Im cpSecYsynthetic peptide Figure 2-7. cpSecY is part of the cpSec complex. The experimental conditions were similar to Figure 2-6. Cross-linking reactions were conducted with 1 mM DSS. A. Crosslinking products on 25 |ig chlorophyll of thylakoid membranes were immimoprecipitated with antibodies against two internal stroma-facing regions of cpSecY (Inl, In2 synthetic peptides) in the presence or absence of the corresponded synthetic peptides (l-2ug/ul serum). AntiSecA immunoprecipitation in the presence of SecY-synthetic-peptide (In2) was used as control. The preimmue sera for both anticpSecY (anti-In2) and antiSecA {Lane 1 and Lane 2 respectively) were also used as controls. B. iOE33 precursor crosslinking products from both the stroma (XSI) and the thylakoid membrane(XMI) were immunoprecipitated with antiLHCP serum. C. Cross-linking products on the 25 ^g chlorophyll of thylakoid membrane were immunoprecipitated with antibodies against the extreme carboxyl terminus of cpSecY in the presence or absence of the cpSecY synthetic peptide. Anti cpSecA immunoprecipitations in the presence or absence of cpSecY synthetic peptide demonstrated the specificity of the peptide effect. The preimmue sera of cpSecY antibody (Pre) was also used as control. Lane 1 contains cross-linking products of 12.5 ^g chlorophyll of thylakoid membrane. The remaining lanes contain immunoprecipitate obtained from 25 ^g chlorophyll of crosslinked thylakoids.

PAGE 49

43 Since the cpSec complex contained cpSecY, we flirther wanted to know if cpSecY played any role in the precursor binding. Antibodies against the C-terminal region of cpSecY inhibited protein transport on the cpSec pathway (Mori et aL, in preparation). After thylakoids were pre-incubated with these antibodies, precursor binding on the thylakoid membranes were significantly reduced. The corresponding antigen reversed the precursor binding reduction (Figure 2-8). Formation of the cpSecA-containing Complex is Pathway-specific It has been shown that precursor proteins transported by the Delta pH pathway and cpSRP pathways do not require cpSecA (Yuan et al. 1994). Therefore these precursors are not e}q>ected to form a cpSecA containing complex. Similar crosslinkmg reactions were conducted with precursors from different pathways (Figure 2-9, Panel A). Several different crosslinking agents were used to ensure that a crossUnking product would be obtained if precursors were in contact with cpSecA. CpSecA-containing complexes were obtained with iOE33 and tPC, both are substrates of the cpSec pathway. In contrast, no cpSecA-containing complexes were obtained with the Delta pH pathway substrate iOE23 or the cpSRP pathway substrate LHCP. However, very little crossUnking was achieved for iOE23, possibly because it does not bind thylakoids tightly. Additional experiments were conducted with Delta pH pathway precursors that do bind to the membrane (Figure 2-9, Panel E). tOE17 is a truncated form of iOE17; DT-17 and DT-23 are precursors that possess a dual targeting (DT) signal peptkie. When the DT-signal peptide is fused to a cpSec pathway passenger protein, the resulting precursors are transported on both the cpSec and the Delta pH pathway. However, fusion proteins contaming Delta pH

PAGE 50

44 Bof Im PI Im Bof TP 1 2 3 4 5 Antigen . . . + + Figure 2-8. iOE33 binding was reduced on anti-cpSecY pre-treated thylakoids. 0.33 ng chlorophyll /fxl of thylakoids were preincubated with 0.8 jig IgG /^l of anti-cpSecY (Cterm.) or preimmune in 3% BSA-IBM buffer in the presence or absence of 15 fxM cpSecY (C-term.) synthetic peptide. The incubations were for one hour in an ice bath. Thylakoid membranes were washed once with IB buffer and used for iOE33 binding assays as describing in methods. Lane TP contains 0.25 ^1 iOE33 translation product. Lanes 1-5 contain 6.25 ^g chlorophyll of thylakoid membranes from the binding assay samples.

PAGE 51

45 DMSO BS^ MBS APDP B O Q Q Q I X-Linldng Q Q Immunoprecipitation Figure 2-9. Complex formation with different precursors. A. Crosslinking studies with a variety of precursors and crosslinking agents. The anticpSecA immunoprecipitation products of the cross-hnking complex from the thylakoid membrane are shown. ImM cross-linkers BS^ (Bis [sulfosuccinimidyl] suberate),or MBS (m-Maleimidobenzoyl-Nhydroxysuccinimide ester), or APDP (N-[4-(p-Azidosalicylamido) butyi]-3'-[2'pyridyldithio] propionamide) were used for the samples in panels as marked. DMSO is the solvent used for preparing the cross-linkers MBS, APDP. tPC is a truncated form of the pPC precursor and is transported by the cpSec pathway (Henry et al. 1997); iOE23 is transported by the Delta pH pathway. LHCP is integrated into the thylakoid membrane by the chloroplast SRP pathway. B. Crosslinking with precursors that bind tightly to thylakoids. tOE17 is a truncated form of iOEl? and is transported exclusively by the Delta pH pathway; DT33 contains a dual targeting signal peptide and is transported by the cpSec and the Delta pH pathway; DTI 7 also contains the dual targeting signal peptide, but is only transported by the Delta pH pathway. Binding assays were as described above and contained apyrase (2 units/75 ^1 assay). DSS was added to the crosslinking reaction at ImM. The cross-lmking complex formed on the thylakoid membrane and the anticpSecA unmunoprecipitation product of the complex are shown in the left and right panels, respectively.

PAGE 52

46 passenger proteins, although readily transported by the Delta pH pathway, are not translocated by the cpSec pathway (Henry et aL 1997). In addition, these later proteins bind tightly to the membrane. As shown in panel B, total membrane crosslinking products were obtained for tOE17, DT17, DT-23 (not shown), and DT33. However, CpSecA-containing crossUnking products were only obtained for iOE33 and DT-33. Thus, only those precursors that can be transported by the cpSec pathway, produced cpSecA-containing complexes. Discussion Transport of proteins on the thylakoidal cpSec pathway requires stromal protein, specifically cpSecA, ATP, and is stimulated by a transmembrane ApH. Protease treatment of thylakoids eliminates their ability to translocate proteins, indicating that thylakoid membrane bound proteins are also involved. With regards to precursor requirements, a Sec-type signal peptide is essential and a compatible mature protein domain is important for efficient transport. Here we have confirmed previous results of Haward et aL (1997), that cpSec transport can occur in two stages: the first is precursor binding to the membrane and formation of a conq)lex with cpSecA. The second step is translocation across the membrane. We have characterized the binding and translocation steps in some detail, determined at which of these steps the above transport requirements are enqjloyed, and examined the stage at which precursor sequences are involved. Our analyses confirm Haward et aL's (1997) observation that iOE33 formed a productive thy lako id-bound intermediate during the transport when apyrase is present to eliminate ATP from the reaction. Furthermore, our results confirm that the intermediate contains cpSecA. We

PAGE 53

47 show here that cpSecY is also present in the complex, providing the first evidence that cpSecY plays a role in cpSecA-dependent protein transport. cpSec precursor binding to the thylakoid membrane requires cpSecA, and accumulation of the bound precursor is resulted from transport inhibition in the absence of ATP. Subsequent transport of the bound precursor requires ATP, but was not stimulated by additional cpSecA. Precursors capable of being transported by the cpSec pathway were crosslinked into the cpSecA contained complex, whereas precursors transported by the Delta pH pathway and SRP pathways were not. As noted by Haward et al. (1997), binding does not require ATP, but occurs maximally in its absence. In our experiments, it was necessary to deplete ATP from assay mixture with apyrase to obtain maximum binding to the membrane. We also tried other methods for arresting the transport and promoting precursor binding such as nonhydrolyzable ATP analogues, ionophores, and azide. But so far, apyrase is the only reagent that could promote precursor binding and the complex formation. The apyrase function for arresting precursor transport is presumed to be its ATPase activity that depletes ATP in the reaction. Boiled apyrase lost its function for stimulating precursor binding and the complex formation (data not shown). The effect seen by removing ATP was a specific one because simply inhibiting the transport by dissipating the pH gradient or adding sodium azide were ineffective in promoting binding and had Uttle effect on binding even when ATP was depleted from the assays. Binding of Sec precursor proOmpA to the E. coli membrane also occurred in the absence of ATP (Cimningham et al. 1989). Bound iOE33 was transported from the bound state into the lumen upon resupply of ATP. It is surprising that a ApH did not stimulate the chase reaction. This could

PAGE 54

48 explain, in part, the relatively low percentage of bound precursor that is subsequently transported. In a typical transport assay, the ApH is responsible for -70% of the transport of iOE33. In E. coli, the last step of proOmpA transport could be achieved by either Sec A related ATP hydrolysis or PMF (proton motive force) alone in the absence of ATP and SecA (Schiebel et al. 1991). For unknown reason, it seems that the bound iOE33 could only be chased into thylakoid lumen with cpSecA related ATP hydrolysis. A ApH actually reduced the chase because in the presence of ionophores, that eliminated the delta pH, the chase was stimulated. A recent report indicates that PMF facilitates deinsertion of SecA from E. coli membrane (Nishiyama et aL 1999). A ApH might undergo similar frinction and result in less chase because release of cpSecA from thylakoid membrane. Chloroplast SecA (cpSecA) facilitates OE33 transport into the thylakoids (Yuan et al. 1994b, Nakai et al. 1994). The fact that most cpSecA is localized in stroma implies that CpSecA functions at early stage of precursor transport which starts in the stroma and ends in the thylakoid lumen. Indeed, cpSecA stimulated precursor binding (Figure 2-2A). The cpSecA was involved in the precursor binding was also suggested by azide treatment which slightly reduced precursor bindmg and subsequent chase. Azide is a characteristic inhibitor of the ATPase activity of SecA (Oliver et aL 1990). Of interesting is that chase of bound precursor into the thylakoid lumen was not stimulated by soluble cpSecA (Figure 2-4). In E. coli, SecA promotes precursor translocation by undergoing ATPdriven cycles of membrane insertion and deinsertion. Each cycle of SecA insertion and deinsertion drives 20-30 aminoacyl residues of the precursor across the membrane (Economou and Wickner 1994). So, complete translocation of a precursor requires

PAGE 55

49 multiple cycles of SecA insertion and deinsertion. It was suggested that after the deinsertion the membrane bound SecA might exchange with soluble SecA or reenter the insertion cycle. From our work, because soluble cpSecA could not stimulate the chase, it seems that cpSecA engaged in the precursor binding/transport maintains contact with the membrane during the precursor chase. This is consistent with the results of Chen et aL (1996) for E. coli SecA. The soluble cpSecA's inability to stimulate the chase also implies that, during transport, cpSecA binds to the thylakoid membrane before precursor does as demonstrated in E. coli system by Cunningham et al. (1989). The boimd precursor could be crosslinked into a high molecular weight conplex. Since the complex formation required cpSecA and the complex contained cpSecA and cpSecY, it is called cpSec conqilex. The detection of such a cpSec complex with bound precursor was an indication that the precursor was engaged in the transport machinery. The idea was further supported by the fact that the precursor binding on the thylakoid membrane was mediated by the protein-protein interactions (Figure 2-5). The results are consistent with those regarding E. coli SecA, that binding between precursor and SecA is very weak in solution, that a strong complex is formed between precursor and SecA on the membrane, and that this complex also contains cpSecY. My results differed from those of Haward et aL (1997) in two respects. First, they obtained a 1:1 crosslinking product between cpSecA and precursor. My crosslinking product was a large migrating product that contains both cpSecA and cpSecY. For the most of my work, crossUnker DSS was used at 1 mM while they used DSS at 0.1 mM or less. A lower concentration of cross-linker might reserve individual cross-linking products which may merge to a larger product with other components when higher concentration cross-linker was used. The

PAGE 56

so reason for higher concentration cross-linker usage in our experiments was that I could have higher yield of cross-Unking product interested. Haward et al. had also obtained a large cross-linking product, but may not show it in their immunoprecipitation. Second, they did not find cpSecY in their complex. Characteristics of their antibodies were not reported. It does not know if their antibodies recognize cpSecY during immunoblotting or immunoprecipitation. In this study, all of antibodies raised against cpSecY immunoprecipitate cpSecY in vitro translation product. The pea C-terminal antibody also recognizes cpSecY during immunoblotting (Mori and Cline, unpublished). Presumably, cpSecY fimctions in thylakoid transport in a manner similar to SecY in bacteria, forming part of the membrane translocon. The present results showing that CpSecY was a con:qx)nent of the crosslinked cpSec complex and provides evidence that cpSecY functions with cpSecA in an analogous manner as the bacterial homologues. Recent results from Mori and Cline (impublished data) demonstrate that antibodies against cpSecY synthetic peptide inhibit protein transport on the cpSec pathway. The cpSecY antibodies inhibited iOE33 binding, indicating that cpSecY was involved in the binding step. iOE33 could either bind to a component associated with cpSecY or bind directly with cpSecY. The antibodies could reduce the binding by disturbing the indirect or direct association between iOE33 and cpSecY. To know the cpSec complex size is important because it will give information about the conplex composition. From the results shown above, the cpSec complex contains at least cpSecA , cpSecY and precursor. CpSecA may work as a dimer. So, the complex could be at least about 300 kD. When the complex was analyzed with SDS-PAGE, it migrated on the gel in a somehow abnormal way. It remains on the top of the separating

PAGE 57

51 gel when a acrylamide concentration higher than 3.5% was used, and it appeared as a smeared band on a 1-16% gradient gel. Under the same conditions, molecular weight markers (67-669 kD) ran as discrete bands. I do not quite understand the con^lexes' behavior on the gels. The cross-linked complex may represent a mixture of multq>le products. In E. coli, the Sec translocase contams more than 9 sub-units (Duong and Wickner 1997). Part of our cross-linking products could contain all the components in the thylakoid transport machine and other parts could contain only several components of the machine. CpSec con^lex formation was precursor specific (Figure 2-9). Our analysis shows that precursors destined for the Delta pH translocation system do not enter into the cpSec complex. Even when Delta precursors bind strongly to the membrane and can be crosslinked to thylakoid membrane proteins, they fail to be crosslinked to cpSecA. Of special interest are chimeric precursors that consist of a dual targeting signal peptide linked to a Delta pH passenger protein. These proteins also fail to be crosslinked to cpSecA. Previous studies have shown that the DT signal peptide is capable of directing eflScient cpSec transport when coupled to a passenger protein that is normally transported on the thylakoidal Sec pathway, but not when linked to Delta pH passenger proteins (Henry et aL 1997). This suggests that these proteins may not be recognized by cpSecA, possibly because they caimot adopt a conpatible loop for the cpSecA system. It has been shown in E. coli that the first 20-30 residues of the mature protein sequence play an important role in transport. When impaired basic residues are introduced into this regron, it poisons translocation. Delta pH precursors do contain significant numbers of basic residues in their N-termini. Support for this notion comes from recent experiments that

PAGE 58

53 In summary, my results are consistent with the model that thylakoidal Sec substrates are committed to the Sec pathway on the membrane and that the initial region of the mature protein as well as the signal peptide plays a critical role in that reaction.

PAGE 59

CHAPTER 3 PROTEIN TARGETING AND TRANSLOCATION ON THE DELTA pH PATHWAY Abstract The Delta pH pathway is one of two systems for protein transport into the thylakoid lumen. This pathway requires only the trans-thylakoid pH gradient to power translocation. A newly constructed precursor protein (tOE17), that uses the Delta pH pathway, binds to the membrane in substantial amount in the absence of the ApH. Binding is productive because bound precursor is transported into the lumen when the ApH is restored. Several observations suggest that binding is due to protein-protein interactions. Protease pretreatment of thylakoids reduced their ability to bind precursor; precursor binding was sensitive to salt concentration; and productive binding was competed by saturating amounts of a Delta pH pathway precursor protein. These results suggest that precursor tOE17 binds to components of the Delta pathway translocation machinery. Transport from the bound state, which we refer to as chase, was very efficient up to 90% of bound precursor could be chased into the thylakoid lumeiL Consistent with the transport requirements, the ApH was the only energy source necessary for the chase reaction. The identification and characterization of a productively bound intermediate in this study provide biochemical tools for detecting the nature and function of components of the Delta pH pathway. Antibodies raised to E. co//-expressed Hcfl06, when pre-bound to thylakoids, specifically inhibited transport on the Delta-pH pathway and reduced the 54

PAGE 60

55 level of productive binding. The results suggest that Hcfl06 functions in early steps of the transport process, possibly as a receptor. Introduction The Delta pH pathway was &st recognized as a distinct transport pathway by the discovery that different subgroups of precursors had different energy and soluble protein requirements for translocation. Specially, transport of precursors 0E17 and OE23 required neither soluble protein factors nor NTPs. Their transport depended entirely on the trans-thylakoid ApH (Cline et al. 1992). A requirement for specific translocation machinery was fiirther demonstrated by precursor competition studies (Cline et al. 1993). In a competition assay with thylakoids, saturating amoimts of iOE23 selectively conqjeted with iOE17 on the same pathway but not with iOE33 on the other pathway. Although the Delta pH pathway was identified years ago, knowledge about its mechanism is quite limited. One reason is that intermediate steps of process had not been identified. Identification of intermediates in other systems directly led to identification of con^nents. The import of protein across the chloroplast envelope is one excellent example (reviewed by Cline and Henry 1996, Schnell 1998). In the presence of low concentration of NTPs (50-100 \iM), protein import into chloroplast is arrested, resulting in bound precursor on the envelope membrane. When the ATP concentration is raised to approximately 1 mM, the bound precursor is translocated into chloroplast. Bound intermediates were then used to identify import machinery components (Schnell and Blobel 1993, Schnell et aL 1994, Kessler et aL 1994, Ma et aL 1996). The ability to an-est productively bound precursor on the thylakoid cpSec pathway (see chapter 1 in this

PAGE 61

56 dissertation and Haward et al. 1997) led to identification of components and precursor elements in the targeting process. Biochemical approaches, such as cross-linking and native gel techniques to identify machinery components have not been successful for the Delta pathway system because intermediates have not been produced. In this study, certain truncated Delta pH pathway precursors were observed to bmd tightly to the thylakoid membrane in the absence of ApH, and the bound precursors were transported into the thylakoids after the ApH was restored. The characteristics of precursor binding and chase process will be described. Materials and Methods Preparation of Radiolabeled tOE17 by in vitro Translation The coding sequence of tOE17 was amplified with the forward primer 5'AATTATGGCGGGCCGCCGCGC-3' and reverse primer 5'GTTTTCCCAGTCACGAC-3' (Nebl212.Pri) using a pGEM7zf plasmid containing maize pOE17 as a template. The PGR product was restricted with Xbal and ligated into Xbal and S/wa/ restricted pGEM 4Z. Capped RNA for tOE17 was produced as described by Cline (1988). RNA was translated m a wheat germ system (Cline et al. 1989) in the presence of [^H]-leucine. Preparation of Other Precursors In vitro transcription plasmids for iOE33, iOE23 and LHCP were described as Cline et al. (1993). Preparation of tOE23, iOE17, iPSAN, DTPC, DT17, DT23, DT33 were described as Henry et al. (1997). Preparation of HyaPC was described as Mori et al. (1998).

PAGE 62

57 Over-expression of tOE17, DT23 and pOE33 in JF. co/i The coding sequence of tOE17 was an^)lified with the forward primer 5'CGTTGCGGGATCCGGCCGCCG-CGCCGTGATCG-3' and reverse primer 5'CCATTATTAGAATTCGCGTCTAGCCTAGCTTGGCG -3' Using the pOE17 plasmid as a template. The PCR product was restricted with BamHI and EcoRI and ligated into appropriately restricted pGEX-2T plasmid. tOE17 was expressed as a glutathione S transferase (GST) fusion protein in E. coli. Expression, purification and digestion of the fusion protein with thrombin followed the BRL conpany instructions. The final tOE17 product after thrombin cleavage was two amino acids different from the in vitro translation product. Instead of the amino acids MA, E. coli-expressed tOE17 has the amino acids GS at its amino terminus. E. coli-expies&ed DT23 and pOE33 were produced as described respectively by Henry et al. (1997) and Cline et al. (1993). Preparation of Chloropiasts, Lysates, Thylakoids and Stroma Pea chloropiasts were isolated from 9-10-day-old seedling; lysates, thylakoids and stromal extract were fM-epared from intact chloropiasts as described by Cline (1991). Thylakoid Protein Transport Assay Transport assays were conducted in 1.5 ml microcentrifuge tubes. In vitro translation product (0.5 to 5 ^1) or 0 to 2 \iM E. coliejq)ressed precursor was incubated with 12.5 fxg chlorophyll of thylakoids or chloroplast lysate in a total volume of 37.5 ^il of import buffer (50 mM Hepes buffer pH 8, 0.33 M sorbitol). Transport reaction was conducted in 70 nE m'^s"' white light for 15 minutes at 25''C. After incubation, thylakoids were recovered by centrifiigation and washed with import buffer. Final membrane samples

PAGE 63

58 were either analyzed directly on SDS-PAGE or treated with 10 thermolysin in 100 ^1 of import buffer (Cline, 1986) and then analyzed by SDS-PAGE and flurography. Precursor Binding and Chase Assays Ten jil [^H] labeled in vitro translated precursor was incubated with 25 jig chlorophyll of thylakoids or chloroplast lysate in the presence of 3 |xM CCCP or 2 units of apyrase for 15 minute at 0"C in a total volume of 75 |j,l of import buffer. The thylakoid membranes were then pelleted at 2500x g, washed with import buffer two times, and divided into two equal portions. One portion was directly analyzed to access the amount of precursor bound to the thylakoid membrane. The second portion was incubated under light for 15 minutes at 25"C in a total of 37.5 |al of chase buffer mixture, which contained -200 \ig protein of stromal extract and ImM DTT. In some assays, thylakoids recovered from binding or chase assays were treated with 10 ^g of thermolysin in 100 ^1 of import buffer (Cline 1986). Preparation of Thermolysinor Proteinase KTreated Thylakoids for use in Binding Assays i Thermolysin-treated thylakoids were prepared as following: 0.5 ^g chlorophyll/|al of thylakoids were incubated with 0-160 \ig thermolysin/ml from a 2 mg/ml stock containing 10 mM CaCli. The incubation was in import buffer on ice and lasted for about 60 minutes. The thylakoid membranes were then recovered by centrifugation and washed twice with import buffer containing 10 mM EDTA and once with stromal extract (containing ~2.5 |ig protein/^1) in import buffer. The washed thylakoids were then resuspended in import buffer at 1 mg chlorophyll/ml. The proteinase K treatedthylakoids were prepared as following: 0.5 ^ig chlorophyll/jil of thylakoids were

PAGE 64

59 incubated with 0-80 protease K /ml from a Img/ml stock. The incubation was in import buffer on ice and lasted for about 60 minutes. PMSF (Phenylmethylsulfonyl Fluoride) was added to 5 mM and the incubation was continued for an additional 10 minutes on ice. Thylakoid membranes were then recovered by centrifiigation and washed twice with unport buffer containing 2 mM PMSF and once with stromal extract (-2.5 l^g/^l) in import buffer. The washed thylakoids were resuspended in import buffer at Img chlorophyll/ml. Analysis of Samples Samples recovered from the assays were subjected to SDS-PAGE and fluorography (Cline 1986). Quantification of the amount of transport, binding or chase was accomplished by scintillation counting of radiolabeled proteins extracted from excised gel bands (CUne 1986). Results Dissipating the ApH across Thylakoids Arrested tOE17 Transport and Resulted in Accumulation of a Transport Intermediate on the Thylakoid Membrane Protein transport into the thylakoid lumen is presumed to start with a targeting event in which the precursor interacts with a transport machinery component. For the Delta pathway, precursor targeting undoubtedly occurs on the thylakoid membrane because soluble components are not required for transport. However, before this study, stable precursor binding to the thylakoids had not been observed. I surveyed a number of precursors, both authentic and recombinant, and found several ones that bound to the thylakoid membrane in the absence of ApH (see below).

PAGE 65

60 tOE17 is an N-terminal truncated form of iOE17 that I constructed as part of another study on targeting determinants (Henry et al. 1997). All thylakoid precursor proteins contain lumen-targeting domains (LTD) which can be divided into four sub-domains: an acidic domain (A), a charged domain (N), a hydrophobic domain (H), and a cleavage domain (C) for proteolytic processing. tOE17 contains only the N, H and C domains. While the H, C domains and the followed mature part are exactly the same as iOE17, the N domain in the fer end of tOE17 contains only MAGRR amino acid residues. According to Henry et al. (1994, 1997), N, H and C domains in the LTD are sufficient for specific precursor targeting and transport. Unexpectedly, truncation of iOE17 produced our most efficient Delta pH pathway transport substrate. Figure 3-1 shows transport assays conducted with tOE17 under varying assay conditions. The assay was started by incubating radiolabeled tOE17 in vitro translation product with the purified thylakoids. Upon transport into the thylakoid lumen, the LTD is removed fi-om the precursor, resulting in the smaller mature 0E17. Figure 3-1 demonstrates that tOE17 transport was inhibited when the ApH across thylakoid membrane was either dissipated by a combination of ionophores, nigericin and valinomycin, or prevented from forming by incubation in the dark with apyrase to deplete ATP. This figure also shows that tOE17 exhibits transport conditions identical to authentic Delta pH pathway precursors, i.e. it occurs in the absence of ATP or stroma, and it was absolutely dependent on the pH gradient. Although tOE17 transport was inhibited by depleting the ApH, a substantial quantity of precursors remained bound to the thylakoid membrane. Bound precursor was degraded by protease treatment of thylakoids, demonstrating that it was exposed on the exterior of

PAGE 66

61 ApH + + + + light + + + + + ATP + + + + ionophores + stroma + + + + + apyrase + + azide + P 1 2 3 4 5 6 P m Protease posttreatment Figure 3-1. tOE17 transport was inhibited by dissipating the ApH, resulting in accumulation of bound precursor on the thylakoid membrane. Ten ^1 [^H]-leucine labeled tOE17 precursor was incubated with 25 )ag chlorophyll of chloroplast lysate or isolated thylakoids (lane 5) in a total of 75 ^1 of import buffer. The assays were conducted under conditions designed to test the effects, on binding and transport, of removing ATP or stromal extract, of dissipating the ApH, or of inhibiting cpSecA with azide. Stromal extract was about 400 ^g of protein per 75 p.! assay, Mg-ATP was added to 5 mM final concentration, the ionophores nigericin and valinomyciti were added to 0.5 \iM and 1 \iM, respectively, from ethanolic stocks. Apyrase treatment was 2 units /75 \il assay. Azide was added to 10 mM final concentration. After incubation at 25°C for 15 minutes the thylakoid membranes were recovered by centrifugation, washed with import buffer two times, and divided into two equal portions in new microfuge tubes. One portion was directly analyzed with SDS-PAGE and fluorography {top panel). The second portion was first treated with protease and then analyzed by SDS-PAGE and flurography (bottom panel). Each lane contained 12.5 ^g chlorophyll of thylakoid membranes. Lane P represents 0.25 ^.1 (5%) precursor added to the assay reaction. The positions of the precursor tOE17 (p) and the mature form (m) are marked.

PAGE 67

62 the thylakoids. The mature 0E17 was resistant to protease treatment confirming its presence in the thylakoid lumen. Bound Precursor is a Productive Intermediate because It can be Chased into the Lumen when the ApH is Restored If the tOE17 binding is physiologically significant, then bound precursor should proceed into the thylakoid lumen once the ApH is restored. I refer to this transport from the bound state as "chase". Binding and chase assays were conducted as shown in Figure 3-2. Radiolabeled precursor was incubated with thylakoids in the dark in the presence of the ionophore CCCP (Carbonylcyanide 3-chlorophenylhydrazone) or with apyrase at 0"C for 15 minutes, during which time tOE17 precursor bound to the thylakoid membrane. Then unbound precursor and ionophore or apyrase were washed away with import buffer and the thylakoids with bound precursor were resuspended in buffer containing stroma and DTT (dithiothreitol) and transferred to 25*'C and white Ught. Under light the bound precursor was chased mto the thylakoids. The time required for total chase was ~8 minutes. About 5.6-9.4% of the added precursor to the assay was bound to the thylakoid membrane and -80% of the bound precursor chased into the lumen. The results indicated that the bound precursor is a productive intermediate on the Deha pH pathway. Other precursors that can be transported on the ApH pathway, most of them made in Cline's laboratory, were also tested in the binding and chase assays. As shown in Figure 3-2 (B), the ability to bind tightly to the thylakoid membrane varied among these precursors. tOE17, DTI 7 and DT23 bound strongly to the thylakoid membrane and a substantial percentage of bound precursor was chased into thylakoids. iOE17 exhibited moderate binding and some bound was chased into thylakoids. HyaPc or iPSAN

PAGE 68

63 Bind Chase A P 1 2 3 4 5 6 Binding with ^^^^i^ « apyrase Binding with CCCP Protease "° + + post-treatment Bind Chase Bind Chase Bind Chas e B P1234 P1234 P1234 iPASNjumH iOEl?! tOE23lCTBHHi iOE23j' dtOE23 — • HyaPC^^^^ dtPC^ dtOE33 — Protease . + . + . + . + + + post-treatment Figure 3-2. Precursor binding-chase experiments. A. Seventy five \ig chlorophyll of thylakoids were incubated with 30 ^l [^H]labeled tOE17 in vitro translation product in darkness in the presence of 3 ^iM CCCP or 6 units of apyrase in a total 225 \i\ of import buffer. The incubation was for about 15 min on ice. The thylakoid membranes were washed with import buffer, divided into 6 portions, and transferred to new tubes. One portion was analyzed directly on SDS-PAGE {lane 1). The second and third portions were treated with thermolysin {lane 3) or mock-buffer {lane 2) and then analyzed on SDS-PAGE. The remaining portions were incubated for chase into the lumen. The chase started with the recovered thylakoids from the binding incubation. The thylakoids were resuspended in 37.5 nl IB containing about 180 |xg protein of stromal extract and ImM DTT (dithiothreitol). The resuspended thylakoids were transferred to 70 nE.m'^s"' white light and incubated for 15 minutes at 25°C. Chase samples were then analyzed directly {lane 4), or treated with thermolysin {lane 6) or mock-buflfer {lane 5) and then analyzed on SDS-PAGE. Each lane was loaded with 12.5 |j.g chlorophyll of thylakoids from each assay. Lane p represents 0.25 \i\ of the precursor added to the assay reaction. B. The assays were conducted as described above with a variety of precursors that can be transported on the Delta pH pathway. Binding assays were in the presence of 3 |j.M CCCP. Lane I, bound precursor on the washed thylakoids; lane 2, thermolysin treated binding samples; lane 3, chase samples; lane 4. thermolysin treated chase samples. Lane p represents 0.25 |xl of the precursor added to the assay reaction.

PAGE 69

64 exhibited substantial binding but the bound HyaPC and iPSAN were not subsequently chased into the lumen. There was hardly any binding for tOE23, iOE23, DT33 and DTPC. The reason that some precursors can bind better than others is not clear. The binding ability appears to be related to both precursor LTD and the mature protein sequence. More information about these precursors can be found in papers published by Henry et al. (1997) and Mori et al. (1998). I have examined several ways of dissipating the ApH m the dark as a means of accumulating bound precursor. These included apyrase treatment to scavenge all traces of ATP, which could be hydrolyzed by proton-pumping CFi/CFo to generate ApH. These also included CCCP treatment. Three jiM CCCP was the optimal concentration for arresting the transport and allowmg chase. Lower CCCP concentration did not completely arrest transport. Higher CCCP concentration resulted in less binding and did not permit chase. CCCP has the advantage of being water soluble, such that it can be washed from the thylakoid membranes. It is possible that high levels of CCCP cannot be adequately removed by washing or, alternatively, that the high level of CCCP conq)romises membrane integrity. The ionophore combination of nigericin and valinomicin resulted in high levels of precursor binding, but the bound precursor was not chased. This is consistent with the fact that nigericin and valinomycin are lipid soluble and probably persist though washing steps. Initially, the binding assay was conducted at room temperature. Subsequent tests indicated that binding under icy temperature was comparable to that at room temperature, resulted in a higher percentage of chase. I found it necessary to eliminate the ApH, even when binding assays were conducted at 0°C. Because transport of some precursor on the Delta pH pathway is so efficient that it occurs

PAGE 70

65 at icy temperatvire (Figure 3-3). Precursor binding and subsequent chase on the Delta pH pathway occxirred in the presence or absence of stromal extract, so stroma was not required for productive binding (data not shown). Chase Requirements for the Productively Bound tOE17 The ApH across the thylakoid membrane is the only energy requirement for protein transport on the ApH pathway. Although I assimied that transport of bound tOE17 occurred on the Delta pH pathway, it was important to examine the chase requirements. A chase requirement assay is reported in Figure 3-4. The chase was inhibited by either nigericin and valinomycin or dark plus apyrase treatment. Apyrase treatment in light did not inhibit the chase. These results indicate that ApH is required for the chase. Buffer containing Mg^ resulted in higher percentage of chase than buffer alone. As expected, ATP did not stimulate the chase. Stromal extract was not required for the chase, but had some stimulation effect. I also examined the chase requirements for DT-23. This precursor has a dual targeting signal peptide, but is only transported on the Delta pH pathway. Bound DT-23 was also transported exclusively by a Delta pH pathway reaction. tOE17 Binding is Due to Protein-Protein Interaction Suggesting that the Bound Precursor be Associating with Some Machinery Component on the Transport Pathway The binding-chase experiment demonstrated that the binding is productive. However, I did not know whether the binding is specific or not. Specific binding should exhibit several characters: (1). the bound precursor should be specifically associated with a specific con^nent, probably a protein component of the transport machinery; (2). the

PAGE 71

66 0°C 25°C p 1 2 3 4 tOE17 tOE23 LHCP ||«Protease post-treatment 0"C 25»C p 1 2 3 4 iOE17 DT17 iOE23 t 1DT23 iOE33 DT33 HyaPC DTPC 0°C 25°C p 1 2 3 4 + + Figure 3-3. Substantial transport could be achieved on the Delta pH pathway at icy temperature for some precursors. Nine ^il in vitro precursor was mixed with 25 chlorophyll of chloroplast lysate, 200 |^g protein of stromal extract and 5 mM ATP in a total 75 nl of import buffer. Incubation was conducted either in icy water (0°C) or 25°C in white light. After incubation for 20 minutes, samples were transferred to an ice bath and 0.5 \iM/\ \iM ionophores nigericin/ valinomycin were added. Thylakoid membranes were recovered by centrifugation, washed with import buffer two times, and divided into two equal portions in new microfuge tubes. One portion was directly analyzed with SDSPAGE and fluorography (Lanes 1 and 3). The second portion was first treated with protease and then analyzed by SDS-PAGE and flurography {Lanes 2 and 4). Each lane contained 12.5 \xg chlorophyll of thylakoid membranes present in each assay. Lane P represents 0.25 ^1 precursor added to the assay reaction.

PAGE 72

67 Chase Conditions 00 1 1 i H W i S £ ^ 3456789 B. mSS<"Sz|^^ I Chase Conditions o J. 4> •§ ^ e . & w i 5 t« w 6 7 8 m E9 < :z < ^ ^ P12345678 B Figure 3-4. Chase requirements for the productively bound tOE17 and DT23. A. 225 chlorophyll of thylakoids were incubated with 90 |il ^[H]-labeled tOE17 in vitro translation product in darkness in the presence of 3 |aM CCCP for about 15 min on ice. The thylakoids were recovered by centrifiigation and washed one time with import buffer. The washed thylakoids were resuspended in import buffer, divided into 9 equal portions and transferred to new tubes. One portion was used to assess the amount of bound precursor {lane 1) — half of the thylakoid membranes was directly resuspended in SDS sample buffer; the other half was treated with protease thermolysin before resuspending in SDS san:q)le buffer. The remainimg portions received further chase treatments under conditions designed to test the effects of ATP, ApH, stromal extract (SE) and Mg-H-. Stromal extract was present at about 375 \ig protein. Mg-Hor Mg-ATP was added to 5 mM final concentration. Nigericin and valinomycin Q^N) were added to 0.5 and 1 ^M, respectively, from an ethanolic stock. Apyrase was added at 2 unit/75 \x\ assay. The chase assay was conducted under 70 ^lE.m'^s"' white light (darkness for lane 8) for 15 min. at 25°C. Finally, half samples were treated with thermolysin (low panel); another half samples were mock-treated with import buffer (upper panel). Lane p contained an aUquot of the precursor added to the binding incubation. B. Experiments were conducted similarly as above but precursor DT23 was used. Samples were treated with thermolysin after the chase.

PAGE 73

68 binding should be satuable; (3). The binding should be competed by precursors known to use the same pathway. I tested if the binding involved a protein-protein interaction (Figure 3-5). tOE17 was incubated with protease-treated thylakoids and assayed for binding or transport. Bound precursor was subsequently analized for chase. Before incubation with precursor, the protease-treated thylakoids were thoroughly washed with buffer containing protease inhibitors in order to eUminate any residual protease from the preparation. Precursor bindmg to the protease-treated thylakoids was substantially reduced compared to control (buffer washed thylakoids). Proteinase K pretreatment had less of an effect on precursor binding than thermolysin treatment. The subsequent chase of any bound precursor on both protease treated thylakoids was totally inhibited mdicating that both protease treatments damaged the transport system. This was also demonstrated by assaying the treated membrane for transport with fresh precursor. It is of interest that thermolysin effects on binding and transport are highly correlated. The nature of the precursor binding was also assessed by urea and sodium carbonate washes. Proteins that are embedded in the lipid phase of a membrane are not extracted by these wash treatments, whereas extrinsic or peripheral proteins that are associated with membranes via protein-protein interaction are removed (Fujiki et al. 1982). All bound precursors on the thylakoids were extracted by the O.IM sodiimi carbonate and most of the bound precursors were extracted with 6M urea washes (data not shown). These results fiirther suggest that the precursors bind directly to membrane proteins under e?q)erimental conditions.

PAGE 74

69 Protease pretreatment of the thvlakoids Thennolysin Proteinase K O ^ t^sb^ O \^ ^
PAGE 75

70 The tOE17 Binding is Salt Sensitive The transit peptides in Delta pH pathway precursor contain a twin-arginine motif that is crucial for the transport pathway selection (Chaddock et al. 1995). One possibility is that the positive charges in the motif may be involved in the interaction between precursor and a putative receptor. If so, then binding should be sensitive to salt concentration. The binding assays were conducted in the presence of increasing concentrations of KCl. As shown in the Figure 3-6, precursor binding in the presence of higher concentration of KCl was decreased as was the subsequent chase. Transport, however, was less influenced by KCl. Transport actually increased slightly from 0-150 mM KCl and decreased at further higher concentration of KCl. Productive Binding can be Competed by Delta pH Precursors If the precursor binding on the thylakoid membrane is an interaction between precursor and a receptor-like component, binding should be saturable and con^tible. To test this, binding of radiolabeled in vitro translated tOE17 was conducted in the presence of increasing concentrations of unlabeled E. co//-produced precursor proteins (Figure 37). The presence of increasing concentration of unlabeled tOE17 precursor resulted in decreased radiolabeled precursor binding, although there remained residual binding that actually increased at high concentration of unlabeled tOE17. Importantly, the amount of productively bound precursor decreased dramatically. Productively-bound precursor was determined as that which chased into the limien upon favorable conditions. From the chase experiment, it is significant that con^etition for productive binding follows a similar inhibition curves as competition for transport of freshly added precursor. Thus,

PAGE 76

71 KCl (mM) A. Binding jj^ *** B. Chase C. Transport Figure 3-6. tOE17 binding in the presence of increasing concentrations of KCl (0500mM). Panel A and panel B are binding-chase assays. Eight tubes, each of which contained 10 ^l ^H-labeled tOE17 in vitro translation product and 9 p,l import buflFer, were preincubated with 1 unit (fil) apyrase for 10 minutes on ice and then mixed with 37.5 nl 0, 100, 200, 300, 400, 600, 800, or 1000 mM KCl in import buffer. Twenty five ^g chlorophyU of thylakids in 17.5|4.1 import buffer, which was preincubated with 1 unit apyrase, was added to each tube. The final incubation volume of each tube was 75 fil and the KCl concentration was 0-500 mM. After 15 minutes incubation on ice the thylakoid membranes in each tube were washed respectively with 0, 50, 100, 150, 200, 300, 400, or 500 mM KCl in import buffer and then washed with import buffer. The washed thylakoid membranes were divided to 2 equal portions and transferred to new tubes. One portion was analyzed directly on SDS-PAGE for binding (panel A). The second portion was incubated for chase. The thylakoids in final chase samples were treated with thermolysin and then analyzed on SDS-PAGE (panel B). Panel C shows transport f fi-eshly added precursor. Chloroplast lysate (12.5 \ig chlorophyll) was mixed with 1.5 ^1 tOE17 translation product in the presence of 0-500 mM KCl in a total 37.5 ^l of import buffer. Lane p represents 0.25 ^1 of the precursor added to the assay reaction. The other lanes in panel A, B and C were loaded with 6.25 \ig chlorophyll of thylakoids from each assay.

PAGE 77

72 20000 1 XI 15000 Binding Unlabeled tOE17(rtM) e Chase 9 s 10000 ^ Transport <::> fV *5 ^ Q u 5000 Binding 0 1 1 1 1 Chase 0 0.25 0.5 1 2 Unlabeled tOE17 Transport 20000 o 15000 S I 10000 Q. " 5000 I" Binding Chase Transport Unlabeled dt23(>iM) P O' C5'
PAGE 78

73 there appears to be two kinds of binding involved. One is productive binding, presumably from an interaction between precursor and transport machinery. Another is nonproductive or non-specific bmding. The amount of non-productive binding varied from experiment to experiment. It became a significant percentage of total binding when E. collproduced precursors were present in the binding assay. This is demonstrated most dramatically in the tOE17 binding saturation experiment (data not shown). Therefor, the non-productive binding may result from precursor aggregation on the thylakoid membrane. It seems apparent to me from Figure 3-7 that the productive binding is competible and the non-productive binding is not. Similar results of competition for labeled tOE17 binding-chase and transport were also obtained with unlabeled DT23 as competitor. pOE33 is transported on the cpSec pathway. E. coli produced pOE33 did not have any effects on tOE17 binding, chase or transport. The above results demonstrate that elevated concentration of tOE17 saturates a membrane component involving in the precursor binding process. In Figure 3-7, it is apparent that in vitro translation of tOE17 produces two products. One is tOE17; a second migrates at the approximately location of mature 0E17. This lower band, which varies in amount with the translation reaction, also binds to the membrane. It is unlikely that it is mOE17 because there is only one methionine in tOE17 that could serve as initiator and because it is competed by DT-23 as well as tOE17 but not pOE33, indicating that it is beiug competed by virtue of the targeting sequence rather than the mature sequence. It is not clear how it is produced from the in vitro translation, but may result from early termination, which has previously been observed with wheat germ translation systems (Mori and Cline, unpubUshed).

PAGE 79

74 Bound Precursor Chase is Competible We fijrther want to know whether there is any con:q)etition for transport from the bound state. The radiolabeled tOE17 was allowed to bind on the thylakoids without competitor. After washing away unbound free precursor the thylakoid membranes with bound precursor were then resuspended in the buffer containing 0 to 2 (xM unlabeled tOE17 and a chase reaction was conducted. As shown in Figure 3-8, chase of bound precursor decreased in the presence of unlabeled precursor. The results indicate that in addition to competition for binding the chase reaction can also be con:^)eted. We have noticed that a significant amovmt of bound precursors was chased into the thylakoids even in the presence of high levels of unlabeled competitor. This did not occur in the competition for transport that was conducted as a control with the CCCP treated thylakoids plus freshly added labeled precursor under the same condition as the chase. tOE17 Binding is Not Reversible One possibility for the above chase competition is that during the chase process the bound radiolabeled precursor is released into solution and re-enters the Delta pH pathway as a soluble precursor. In this case, the observed competition for chase would be equivalent to competition for transport of free precursors. To clarify this issue, a different binding con:q)etition experiment was conducted to assess the reversibility of binding (Figiire 3-9). In the above binding competition tests, chemical amount of E. coli. produced unlabeled precursors were incubated first with thylakoids and the radiolabeled in vitro translation precursors were added afterwards. If radio labeled precursors were

PAGE 80

75 E.coli produced ^H-tOE17 (jiM) E.coli produced 'H-tOElT (jiM) ^^V V V 1 2 3 4 5 6 7 Chase Competition Transport Competition Figure 3-8. Bound precursor chase competition. In the left panel, 1 12.5 \i% chlorophyll of thylakoids were incubated with 45 ^il ^Hlabeled tOE17 in vitro translation product in dark in the presence of 3 CCCP. After 15 minutes incubation on ice, the thylakoids were recovered by centrifiigation and washed with import buffer and divided into 9 equal portions and transferred to new tubes. For checking precursor binding, one portion was directly analyzed with SDS-PAGE (lane 1); second portion was treated with protease thermolysin, and then analyzed with SDS-PAGE (lane 2). The thylakoid membranes in the remaining portions (lane 3-9) were resuspended in buffer containing increasing concentrations of unlabeled E coli produced tOEl? and incubated under chase conditions. Final chase samples were treated with thermolysin. Transport assay controls are presented in the right panel. 12.5 (xg chlorophyll of thylakoids, which were pre-treated with 3 |iM CCCP and buffer-washed, were mixed with buffer containing 1 ^1 ^Hlabeled tOE17 in vitro translation product and increasing concentrations of unlabeled E coli produced tOE17 and incubated under chase conditions. Final transport samples were treated with thermolysin. Lane P contains an aUquot of the precursor added to assays. The remainmg lanes in both panels were loaded with 6.25 jig chlorophyll of thylakoids from each assay.

PAGE 81

76 Uniabeled precursor ( ^M) in binding 1000 0 -I 1 i 1 1 0 0.25 0.5 1 2 Competitor concentration (micro morlar) Figure 3-9. Effect of unlabeled precursor on the binding of preloaded radiolabeled tOE17 in vitro translation product. Ten \il radiolabeled tOEl? was incubated with 25 |a,g chlorophyll of thylakoids for 5 min, then 0-2 jxM unlabeled E. coli produced precursor (tOEl?) was added and the incubation continued for 15 minutes. After washing with import buffer, the membranes were transfer to new tubes. Half of each sample was analyzed directly for the amount of precursor bound to the membrane; remaining half of each sample was incubated for chase in a buffer containing stroma and DTT.

PAGE 82

77 incubated first with the thylakoids, its binding was not competed by the added unlabeled precursor. However, the subsequent chase of the bound radiolabeled precursor was decreased. The results demonstrate that the precursor binding is quite strong and hardly replaced. Also, the results further confirm that there is real competition at chase step, though the competition occurs between the bound precursors. Hcfl06 is involved in the tO£17 binding Hcfl06 was identified in maize as a component of the Delta pH pathway by both in vivo and in vitro methods (Voelker and Barkan 1995, Settles et al. 1997), Tha4 is a maize homologue of Hcfl06. a cDNA for psTha4, a pea homologue of Tha4, was recently isolated in Cline's laboratory. Antibodies against maize Hcfl06 or psTha4 were prepared (Mori et al., in preparation). The effects of antibodies against maize Hcf 106, pea psTha4 and pea cpSecY, on tOE17 binding-chase and transport to pea thylakoids, were examined. Thylakoids were pre-incubated with either antiserum IgG or preimmune serum IgG. After washing, the thylakoids were assayed for binding-chase and transport of Unlabeled tOE17. As shown in Figure 3-10, Hcfl06 and psTha4 antibodies inhibited tOE17 transport and chase, indicating Hcfl06 and psTha4 ftmction on the Delta pH pathway. The inhibitions caused by antibody against Hcfl06 could be reversed by antigen Hcfl06 protein. Antibody against cpSecY, which plays an essential role on the cpSec pathway, did not have any inhibition. I have noticed that although both Hcfl06 and psTha4 antibodies specifically inhibited tOE17 transport and chase, only Hcfl06 antibody reduced the binding. Preliminary experiments indicated that the bound tOE17 could form

PAGE 83

78 A B P12345 6789 P 1 2 3 4 5 Chase Chase Transport mm Transport ' Antigen + + Figiire 3-10. tOEl? binding-chase and transport to the thylakoids pre-treated with Hcfl06, psTha4 or cpSecY antibodies. In A panels, 0.33 \ig chlorophyll Z^il of thylakoids were pre-incubated with 0.5 or 1.0 ^.g IgG /|j,1 of immune or preimmune in 3% BSA-IBM buflfer. The incubations were for one hour in an ice bath. Thylakoid membranes were washed once with IB buffer and used for binding-chase and transport assays as describing in methods. In B panels, thylakoids were pre-incubated with 0.8 ^g IgG of antiHcfl06 or preimmime in 3% BSA-IBM buffer in the presence or absence of 15 )xM Hcfl06 antigen. Following steps as A panels. For both A and B panels, Lane P contains 0.25 ^1 tOEl? in vitro translation product. The remaining lanes contain 6.25 \ig chlorophyll of thylakoid membranes from the assay samples.

PAGE 84

79 a complex with Hcfl06. tOE17 was cross-linked on the thylakoid membrane. AntiHcfl06 prevented the formation of cross-linking products. The crossUnkmg products were immunoprecipitated with antibody against Hcfl06. These results suggest that Hcfl06 fimction in the precursor binding process, possibly as a receptor. Discussion For the first time, I show here that a typical Delta pH pathway precursor accumulated on the thylakoid membrane when the ApH was dissipated. The bound precursors were transported into the thylakoids when the ApH was restored. The construction of precursor tOE17 allows me to successfully demonstrate that transport on the Delta pH pathway occurs in two steps, binding and then translocation. I think that the binding step represents the targeting reaction on the Delta pH pathway. The tOE17 transit peptide only has N, H and C domains with the N domain containing MAGRR residues. Nevertheless, tOE17 is a typical substrate for the Delta pH pathway. Its transport does not require any soluble components from the chloroplast stroma and the ApH across the thylakoid membrane is the only energy requirement. Its transport also was competible with native precursor iOE23 from the Delta pH pathway (date not shown) and its transport was totally inhibited by Hcfl06 antibody. Of interest, the first precursor observed to exhibit productive binding is DT23. It has a dual targeting transit peptide but only can be transported by the Delta pH pathway (Henry et al. 1997). In the presence of ionophores DT23 transport was inhibited and substantial precursor remained bound to the thylakoid membrane. Because the DT transit peptide can target a cpSec pathway passenger protein transport on both Delta pH and cpSec pathways and can only target a Delta pH pathway

PAGE 85

80 passenger on Delta pH pathway (Henry et al. 1997), it was thought that bound DT23 was stuck in the cpSec machinery. At that time no Delta pH pathway precursor had been found binding tightly to the thylakoid membrane. However, further experiment indicated that the bound DT23 was on the Delta pH pathway and was not stuck in Sec machinery (data not shown). Furthemore the bound DT23 was subsequently transported on the Delta pH pathway and competed by the Delta pH pathway precursor. All the experiments shown above for tOE17 were first tried with DT23 with similar results. We also found that native precursor iOElT behaved the same in those experiments mentioned above, although the binding was much less. Why tOEl? can bind much tighter than the nature precursor on the thylakoid membrane is not clear. All of tightly bound precursors tOE17, DT23 and DTI 7 have a short transit peptide. However, tOE23 that also has a truncated (short) LTD similar to tOE17 does not bind strongly. The longer transit peptide in nature precursor has an A domain which function is unknown. Precursor binding was sensitive to KCl concentration, indicating that an ionic interaction is involved. All Delta pH pathway precursor contain a twin-arginine motif, which is crucial for targeting the precursor into the pathway (Chaddock et al. 1995). It is highly possible that the RR is used for precursor specific binding. However, without the RR, a truncated precursor named KK17 still boimd on the thylakoid membrane, although it was not subsequently chased into the limien (data not shown). This suggests that RR may not be involved in the binding and its role may be played at translocation step. Besides the twin-arginine, other domains in the transit peptide are also required for the targeting (Chaddock et al. 1995, Henry et al. 1997). Therefore, precursor binding could involve multiple components on the thylakoid membrane. Bound tOE17 could be cross-

PAGE 86

SI linked to thylakoid proteins and the cross-linked complexes could be immunoprecipitated with Hcfl06 antibody. Comparing the cross-linking and immunoprecipitation products from bound tOE17 and KK17 may give insight into whether or not RR specifically interacts with some components on the thylakoid membrane. The binding competition tests in Figure 3-7 demonstrated that the binding was competible, implying that a saturable component of the transport machinery is involved in binding. However, when binding was conducted with increasing concentrations of Unlabeled E. co/;-produced tOE17 the total binding mcreased lineally with higher precursor usage. While the binding of E. co//-produced tOE17 is not satuable, the subsequent chase exhibited saturation. The direct transport also saturable and displayed the same kinetics (data not shown). The chase competition was unexpected. It is not likely but possible that imder "chase conditions" the bound precursor is released into the solution and then reenters the transport pathway. If this is true, then transport of bound precursor cannot be considered to be chased from an intermediate step. The chase is considered to be a process in which the bound precursor directly transfers to the translocon from the binding site and is translocated into the lumen. In this case, the observed chase competition would actually be con^etition for a fiiU transport reaction. Several methods, including conducting chase under different volumes of buffer, were tested to determine whether the chase was achieved directly. The results were inconclusive. However, we noticed that unlike the transport competition control a significant amount of boimd precursor transported into the thylakoids regardless of how high the concentration of competitor was (Figure 3-8). This is evidence that the observed chase is different from the normal transport. The binding is considered to be an interaction between precursor and a

PAGE 87

82 receptor. The subsequent chase should be directly achieved by shift of the bound precursor from the receptor to an associated translocoiL Then, any con^)etition occurring at this step must result from multiple receptors sharing a common translocon. Preliminary crosslinking data from CUne's laboratory (Mori and Cline, unpubUshed data) indicates that Hcfl06 exists in thylakoids as a multimer. If Hcfl06 fimctions as a receptor, competition for chase may reflect the existence of an Hcfl06 multimer that serves as some sort of antenna receptor conq)lex. The Delta pH pathway is a very novel system considering that the ApH across thylakoids is the only energy requirement. It was originally thought to be a unique pathway of chloroplasts. Now its homologues have been found in bacteria (Settles and Martienssen 1998). Before this study, no Delta pH pathway precursors were foimd to bind tightly to the thylakoid membrane, making identification and purification of components very difficult. The binding-chase techniques developed in this study should provide an important tool to isolate machinery components and help understand the transport mechanism.

PAGE 88

CHAPTER 4 SUMMARY AND CONCLUSIONS The first part of my research is about protein transport on the cpSec pathway. Advancing the understanding about the mechanism is achieved by dissecting the transport process into targeting and translocation stages and the related characterization of each stage. Productive binding with precursors to the thylakoid membrane was stimulated with cpSecA and occurred when transport was inhibited by removing ATP from the assay with apyrase. cpSecA may function as a determinant for transport pathway selection as only the precursors using the cpSec pathway can form an complex with it. cpSecY was found to be another machinery component associated with bound precursor, providing the first evidence that cpSecY is functioning on cpSec transport pathway. Subsequent translocation of bound precursor required ATP, but was not stimulated by additional cpSecA. cpSecA had been speculated to be the Sec pathway receptor, given the fact that it is the only soluble component required for the transport on the cpSec pathway. It would be logical that cpSecA interacts with precursor in the stroma and targets the precursor to the translocon in the thylakoid membrane. However, no such interaction was detected. Our results support the notion that precursor commitment to the thylakoid cpSec pathway occurs on the membrane, rather than in the soluble stroma. Since the first detectable interaction between precursor and components of the cpSec pathway machinery was on the membrane, we assume that the receptor residences on the membrane. 83

PAGE 89

84 The second part of my research is about protein transport on the Delta pH pathway. This pathway is unique for its special energy requirement and has been a recent focus of research in thylakoid protein transport. However, for years, knowledge about the mechanisms involved in this system has been quite limited. A biochemical approach such as cross-linking and native gel techniques to identify the machinery components had been difficult for the system since the routinely used precursors did not bind tightly to the thylakoid membrane. A newly constructed precursor protein tOE17 allows me to develop the technique of dissecting the whole transport into binding (targeting) and chase (translocation) steps. tOE17 binds to the membrane m substantial amount in the absence of the ApH and the bound precursor is chased into the lumen when the ApH is restored. Several observations suggest that binding is due to protein-protein interaction. Productive bmding was competed by saturating amounts of a ApH pathway precursor protein, suggesting that machinery components from the Delta pH pathway are involved. Consisting with the transport requirement, the ApH was the only energy source used for chase. Interestingly, chase of the bound precursor could be conq)eted with unlabeled precursor. Hcfl06 and Tha4 are two components identified recently. Both maize Hcfl06 antibody and pea Tha4 antibody specifically inhibited tOE17 transport and chase to the pea thylakoids. However, only antibody raised against Hcfl06 reduced tOEl? binding to the thylakoid membranes. Given the fact that most part of the protein is facing the stroma, Hcfl06 may fiinction as a receptor.

PAGE 90

REFERENCES Abad MS, Clark SE, Lamppa GK. 1989. Properties of a chloroplast enzyme that cleaves the chlorophyll a/b binding protein precursor. Plant Physiol. 90: 11 7-24. Akita M, Sasaki S, Matsuyama S, Mizushima S. 1990. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in E. coll J. Biol. Chem. 265:8164-69. Akiyama Y, Ito K. 1985. The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. EMBOJ. 4:3351-56. Anderson CM, Gray J. 1991. Cleavage of the precursor of pea chloroplast cytochrome f by leader peptidase from Escherichia coli. FEBS Lett. 280:383-86. Bassham DC, Bartling D, Mould RM, Dunbar B, Weisbeek P, Herrmarm RG, Robinson C. 1991. Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins. J Biol Chem. 266:23606-10. Berghoser J, Kamauchov I, Herrmann RG, Klosgen RB. 1995. Isolation and characterization of a cDNA encoding the secA protein from spinach chloroplasts. J. Biol. Chem. 270:18341^6. Bogsch E, Brink S, Robinson C. 1997. Pathway specificity for a delta pH-dependent precursor thylakoid lumen protein is governed by a Sec-avoidance' motif in the transfer peptide and a 'Secincompatible' mature protein. EMBOJ. 16:3851-59. Bogsch EG, Sargent F, Stanley NR, Berks BC, Robinson C, Palmer T. 1998. An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J. Biol. Chem. 273:18003-06. Brundage L, Hendrick J, Schiebel E, Driessen A, Wickner W. 1990. The purified E. Coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell 62:649-657. Caliebe A, Grimm R, Kaiser G, Lubeck. J, Soli J, Heins L. 1997. The chloroplastic protein import machinery contains a Rieske-type iron-sulfiir cluster and a mononuclear iron-binding protein. EMBOJ. 716:7342-7350. 85

PAGE 91

86 Clark SA, Theg SM. 1997. A folded protein can be transported across the chloroplast envelope and thylakoid membranes. Mol. Biol. Cell 8:923-34. Chaddock AM, Mant A, Kamauchov I, Brink S, Herrmann RG, Klosgen RB, Robinson C. 1995. A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta-pH-dependent thylakoidal protein translocase. EMBO J. 14:2715-22. Chen X, Xu H, Tai PC. 1996. A significant fraction of functional SecA is permanently embedded in the membrane. SecA cycling on and off the membrane is not essential during protein translocation, y 5/0/. Chem. 271:29698-29706. Chua N-H, Schmidt GW. 1978. Post-translational transport mto intact chloroplasts of a precursor to the small subunit of riburose-1, 5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 75:61 10-61 14. Chua N-H, Schmidt GW. 1979. Transport of proteins into mitochondria and chloroplasts. J. Ce//5/o/. 81:461-483. Chitnis PR, Nechushtai R, Thomber JP. 1987. Insertion of the precursor of the lightharvesting chlorophyll a/b-protein into the thylakoids requires the presence of a developmentally regulated stromal fector. Plant Mol. Biol. 10:3-1 1 . Cline K, WernerWashbume M, Lubben TH, Keegstra K. 1985. Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J. Biol. Chem. 260:3691-96. Cline, K. 1986. Import of proteins into chloroplasts: Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates. J. Biol. Chem. 261:1480414810[Abstract]. Clme K, Fulsom DR, Viitanen PV. 1989. An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited. J. Biol. Chem. 264:14225-32. Cline K, Ettinger WF, Theg SM. 1992. Protein-specific energy requirements for protein transport across or into thylakoid membranes. J. Biol. Chem. 267:2688-96. Cline K, Henry R, Li CJ, Yuan JG. 1993. Multiple pathways for protein transport into or across the thylakoid membrane. EMBO J. 12:4105-14. Cline K, Henry R. 1996. Import and routing of nucleus-encoded chloroplast proteins. Annu. Rev. Cell Dev. Biol.\2A-26. Creighton AM, Hulford A, Mant A, Robinson D, Robinson C. 1995. A monomeric, tightly folded stromal intermediate on the delta pH-dependent thylakoid protein transport pathway. J. Biol. Chem. 270:1663-69.

PAGE 92

87 Cunningham K, Lill R, Crooke E, Rice M, Moore K, Wickner W, Oliver D. 1989. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOn:q)A. EMBOJ. 8:955-959. de Boer AD, Weisbeek PJ. 1991. Chloroplast protein topogenesis: import, sorting and assembly. Biochim. Biophys. Acta 1071:221-53. Dobberstein B, Blobel G, Chua, N-H. 1977. In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-l,5-bisphosphate carboxylase of Chlamydomonas reinhardtu. Proc. Natl. Acad. Sci. USA 74:1082-1085. Duong F, Wickner W. 1997. Distinct catalytic roles of the Sec YE, SecG and SecDFyajC subunitsof preprotein translocase holoenzyme. £A/fiO J. 16:2756-68. Economou A, Wickner W. 1994. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell 78:835-843. Ento T, Kawakami M, Gogo A, America T, Weisbeek P, NaKai M. 1994. Chloroplast protein import: chloroplast envelopes and thylakoids have different abilities to unfold proteins. Eur. J. Biochem. 225:403-409. Fikes JD, Bassford PJ Jr. 1989. Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide. J Bacterial. 171:402-409. Fincher V, McCaflfery M, Cline K. 1998. Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway. FEBS Letters 423 :66-70. Friedman AL, Keegstra K. 1989. Chloroplast protein import: quantitative analysis of precursor binding. Plant Physiol. 89:993-99. Haehnel W, Jansen T, Gause K, Klosgen RB, Stahl B, et al. 1994. Electron transfer from plastocyanin to photosystem I. EMBO J. 1 : 1028-38. Hageman J, Baecke C, Ebskamp M, Pilon R, Smeekens S, Weisbeek P. 1990. Protein import into and sorting inside the chloroplast are independent processes. Plant Cell 2:479-94. Halpin C, Elderfield PD, James HE, Zimmerman R, Dunbar B, Robinson C. 1989. The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical £M50 J. 8:3917-21. Hand JM, Szabo LJ, Vasconcelos AC, Cashmore AR. 1989. The transit peptide of a chloroplast thylakoid membrane protein is fimctionlly equivalent to a stromal-targeting sequence. £:MS0 J. 8:3195-3206.

PAGE 93

88 Hartl F, Lecker S, Schiebel E, Hendrick J, Wickner W. 1990. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. Coli plasma membrane. Cell 63:269-279. Haward SR, Napier JA, Gray JC. 1997. Chloroplast SecA functions as a membraneassociated component of the Sec-like protein translocase of pea chloroplasts. European Journal of Biochemistry. 248:724-730. Henry R, Carrigan M, McCafifery M, Ma XY, Cline K. 1997. Targeting determinants and proposed evolutionary basis for the Sec and delta-pH protein transport systems in chloroplast thylakoid membranes, y. Cell Biol. 136:823-32. Hinnah SC, Hill K, Wagner R, SchUcher T, Soil J. 1997. Reconstitution of a chloroplast protein import channel. EMBOJ. 16:7351-7360. Hirsch S, Muckel E, Heemeyer F, von Heijne G, Soil J. 1994. A receptor con^nent of the chloroplast protein translocation machinery. Science 266: 1989-92. Hofifinan NE, Franklin AE. 1994. Evidence for a stromal GTP requirement for the integration of a chlorophyll a/b-bmding polypeptide into thylakoid membranes. Plant Physiol. 105:295-304. Hulford A, Hazell L, Mould RM, Robinson C. 1994. Two distinct mechanisms for the translocation of proteins across the thylakoid membrane, one requiring the presence of a stromal protein fector and nucleotide triphosphates. J. Biol. Chem. 269:325 1-56. Hynds PJ, Robinson D, Robinson C. 1998. The Sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane. J. Biol. Chem. 273:34868-34874. Keegstra K, Cline K. 1999. Protein import and routing systems of chloroplasts. Plant Cell. In press. Kessler F, Blobel G, Patel HA, Schnell DJ. 1994. Identification of two GTP-binding proteins in the chloroplast protein import machinery. Science 266:1035-39. Kessler F, Blobel G. 1996. Interaction of the protein import and folding machineries in the chloroplast. Proc. Natl. Acad Sci. USA 93:7684-89. Kim SJ, Robinson D, Robinson C. 1996. An Arabidopsis thaliana cDNA encoding PS 11X, a 4.1 kDa component of photosystem II: a bipartite presequence mediates SecA/delta pH-independent targeting into thylakoids. FEBS Lett. 390:175-78. Kirwin PM, Meadows JW, Shackleton JB, Musgrove JE, Elderfield PD, et al.l989. ATPdependent import of a lumenal protein by isolated thylakoid vesicles. EMBO J. 8:225155.

PAGE 94

89 Klimyuk, VI, Persello-Cartieaux F, Havaux M, Contard-David P, Schuenemann D, Meiherhoff K, Gouet P, Jones JDG, Hoffitian NE, Nussaume L. 1999. A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting. Plant Ce//, 11:87-99. Knott TO, Robinson C. 1994. The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane. J. Biol. C/ieAw. 26911:7843^6. Ko K, Cashmore AR. 1989. Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving proteia EMBO J. 8:3187-94. Kouranov A, Schnell DJ. 1997: Analysis of the interactions of preproteins with the import machinery over the course of protem import into chloroplasts. J. Cell Biol. 139: 1677-1685. Kuhn A, Kiefer D, Kohne C, Zhu H-Y, Tschantz WR, Dalbey RE. 1994. Evidence for a loop-like insertion mechanism of pro-Omp A into the inner membrane of Escherichia coli. Eur. J. Biochem. 226:891-897. Laidler V, Chaddock AM, Knott TO, Walker D, Robinson C. 1995. A SecY homolog in Arabadopsis thaliana. J Biol. Chem. 270: 1 76641 7667. Lamppa GK. 1988. The Chlorophyll a/b-binding protein inserts into the thylakoids independent of its cognate transit peptide. J. Biol. Chem. 263:14996-14999. Lorkovic ZJ, Schroser WP, Pakrasi HB, Irrgang KD, Herrmann RG, OehnuUer R. 1995. Molecular characterization of PsbW, a nuclear-encoded conqwnent of the photosystem 11 reaction center con:q)lex in spinach. Proc. Natl. Acad. Sci. USA 92:8930-34. Ma YK, Kouranov A, LaSala SE, Schnell DJ. 1996. Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope. J. Cell Biol. 134:315-27. Meadows JW, Robinson C. 1991. The foil precursor of the 33-kDa oxygen-evolving complex protein of wheat is exported by Escherichia coU and processed to the mature size. Plant Mol. Biol. 17:1241-43. Meyer T H, M^netret J F, Breitling R, Miller K R, Akey CW, Rapoport T A. 1999. The bacterial SecY/E translocation complex forms channel-like structures similar to those of the eukaryotic Sec61p complex. J. Mol. Biol. 285:1789-800.

PAGE 95

90 Michl D, Robinson C, Shackleton JB, Herrmann RG, Klosgen RB. 1994. Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway. EMBO J 1 3 : 1 3 1 0-1 7. Mizushima S, Tokuda H. 1990. In vitro translocation of bacterial secretory proteins and energy requirements. J. Bioenerg. Biomembr. 22:389-99. Review. Mori H, Cline K. 1998. A signal peptide that directs non-Sec transport in bacteria also directs efficient and exclusive transport on the thylakoid Delta pH pathway. J. Biol. Chem. 273:11405-11408. Mori H, Summer L, Ma X, Cline K. 1999. Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways. (To be submitted). Morin XK, Soli J. 1997. Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery. Planta 201:119-127. Nilsson R, Brunner J, Hoffman NE, van Wijk KJ. 1999. Interactions of ribosome nascent chain complexes of the chloroplast-encoded Dl thylakoid membrane protein with cpSRP54£MfiOJ. 18:733-742. Nishiyama K-I, Fukuda A, Morita K, Tokuda H. 1999. Membrane deinsertion of SecA imderlying proton motive force-dependent stimolation of protein translocation. EMBO J. 18:1049-1058. Nohara T, Asai T, Nakai M, Sugiura M, Endo T. 1996. Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway. Biochem. Biophys. Res. Commun. 224:474—78. OUver DB, Cabelli RJ, Dolan KM, Jarosik GP. 1990. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc. Natl. Acad. Sci. USA 87:8227-31. Olsen LJ, Keegstra K. 1992. The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. J. Biol. Chem. 267:433-39. Osborne RS, Silhavy TJ. 1993. PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains. EMBO J. 12:3391-3398. Payan LA, Chne K. 1991. A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein. J. Cell Biol. 1 12: 603-13. Pain D, Blobel G. 1987. Protein import in chloroplasts requires a chloroplast ATPase. Proc. Natl. Acad. Sci. USA 84:3288-92.

PAGE 96

91 Perry SE, Keegstra K. 1994. Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell 6:93-105. Pilon M, De Boer AD, Knols SL, Koppelman MHGM, Van der Graaf RM, De Kruijff B, Weisbeek PJ. 1990. Expression in Escherichia coli and purification of a translocationconqjetent precursor of the chloroplast protein ferredoxin J. Biol. Chem. 265:3358-3361. Pugsley A. 1993. The complete general secretory pathway in gram-negative bacteria Microbiol Rev. 57:50-108. Puziss JW, Fikes JD, Bassford PJ Jr. 1989. A analysis of mutational alterations in the hydrophihc segment of the maltose-binding protein signal peptide. J Bacteriol. 171:23022311. • ' . Rapoport TA, Jungnickel B, Kutay U. 1996. Protein Transport Across the Eukaryotic Endoplasmic Reticulum and Bacterial Inner Membranes Annu. Rev. Biochem. 65:271-303. Robinson C, Ellis RJ. 1984. Transport of proteins into chloroplasts: partial purification of a chloroplast protease involved in the processing of imported precursor polypeptides. Eur. J. Biochem. 142:337-42. Robinson C, Klosgen RB. 1994. Targeting of proteins into and across the thylakoid membrane: a multitude of mechanisms. Plant Mol. Biol. 26:15-24. Robinson D, Kamauchov 1, Herrmann RG, Klosgen RB, Robinson C. 1996. Proteasesensitive thylakoidal import machinery for the Sec-, delta pHand signal recognition particle-dependent protein targeting pathways, but not for CFqII integration. Plant J. 10:149-55. Robinson C, Hynds PJ, Robinson D, Mant A. 1998. Multiple pathways for the targeting of thylakoid proteins in chloroplasts. Plant Molecular Biology 38:209-221. Sargent F, Bogsch EG, Stanley NR, Wexler M, Robinson C, Berks BC, Palmer T. 1998. Overlapping functions of components of a bacterial Sec-independent protein export pathway fMBO J. 17:3640-3650. Shaw AS, Rottier PJM, Rose JK. 1988. Evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 85:7592-7596. Schiebel E, Driessen AJM, Hartl F-U, Wickner W. 1991. A^ilT and ATP fiinction at different Steps of the catalytic cycle of preprotein translocase. Cell 64:927-939. Schnell DJ, Blobel G. 1993. Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelop contact sites. J. Cell Biol. 120:103-15.

PAGE 97

92 Schnell DJ, Kessler F, Blobel G. 1994. Isolation of components of the chloroplast protein import machinery. Science 266:1007-12. Schnell DJ. 1998. Protein targeting to the thylakoid membrane. Annu. Rev. Plant Physiol. Plant Mol Biol 49:97-126. Schuenemann D, Gupta S, Persello-Cartieaux F, KUmyuk VI, Jones JDG, Nussaume L, Hoffinan NE. 1998. A novel signal recognition particle targets light harvesting proteins to the thylakoid membranes. Proc. Natl Acad. Sci. USA 95:10312-10316. Settles AM, Yonetani A, Baron A, Bush DR, Cline K, Martienssen R. 1997. Secindependent protein translocation by the Maize Hcfl06 Protein. Science 278:1467-1470 Settles AM, Martienssen R. 1998. Old and new pathways of protein export in chloroplasts and bacteria. Trends Cell Biol. 8:494-501. Review. Seidler A, Michel H. 1990. Expression in Escherichia coU of the psbO gene encoding the 33 kD protein of the oxygen-evolving complex from spinac/j. EMBO J. 9: 1 743-48. Smeekens S, Bauerle C, Hageman J, Keegstra K, Weisbeek P. 1986. The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell. 46:365-75. Soil J, Tien R. 1998. Protein translocation into and across the chloroplastic envelop membranes. Plant Molecular Biology 38:191-207. Sugiura M, Hirose T, Sugita M. 1998. Evolution and mechanism of translation in chloroplasts. Annu. Rev. Genet. 32:437-459. Swidersky UE et al. 1992. Biochemical analysis of the biogenesis and fimction of the Escherichia coli export factor SecY. Eur. J. Biochem. 207:803-8 11. Swidersky UE, Hofl&chulte HK, Muller M. 1990. Determinants of membrane-targeting and transmembrane translocation during bacterial protein export. EMBO J. 9:1777-1785. Theg SM, Bauerle C, Olsen LJ, Selman BR, Keegstra K. 1989. Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem. 264:6730-36. Tranel PJ, Froehlich J, Goyal A, Keegstra K. 1995. A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway. EMBO J. 14:2436-^6. VanderVere PS, Bennett TM, Oblong JE, Lamppa GK. 1995. A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases. Proc. Natl. Acad. Sci. USA 92:7177-81.

PAGE 98

93 Viitanen PV, Doran ER, Dunsmuir P. 1988. What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein? J. Biol. Chem. 263: 15000-15007. Voelker R, Barkan A. 1995. Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid. EMBO J. 14:3905-14. Voelker R, Mendel-Hartvig J, Barkan A. 1997. Transposon-disruption of a maize nuclear gene, thai, encodmg a chloroplast SecA homologue: in vivo role of cp-SecA in thylakoid protein targeting. Genetics 145:467-78. von Heijne G, Steppuhn J, Herrmann RG. 1989. Domain structure of mitochondrial and chloroplast targeting peptides. Eur. J. Biochem. 180:535—45. von Heijne G, Nishikawa K. 1991. Chloroplast transit peptides: the perfect random coil? FEBSLett. 278(l):l-3 Waegemann K, Paulsen H, Soli J. 1990. Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import conpetence. FEBS Lett. 261 :89-92. Watanabe P, Blobel G. 1989. Site-specific antibodies against the PrLA (SecY) protein of Escherichia coh inhibit protein export by interfering with plasma membrane binding of preproteins. Proc Natl Acad Sci USA 86: 1 8951 899. Weisbeek P, Hageman J, De Boer D, Pilon R, Smeekens S. 1989. Inqx)rt of proteins into the chloroplast lumen. J. Cell Sci. Suppl. 1 1 : 199-223. Weiner JH, BUous PT, Shaw GM, Lubitz SP, Frost L, Thomas GH, Cole JA, Turner RJ. 1998. A novel and ubiquitous system for membrane targeting and secretion of cofectorcontaining proteins. Cell 93:93-101. Wexler Margaret, Bogsch Erik G, KlSsgen Ralf Bemd, Palmer Tracy, Robinson Colin, Berks Ben C. 1998. Targeting signals for a bacterial Sec-independent export system direct plant thylakoid import by the ApH pathway, Febs Letters 431:339-342. Yuan J, Cline K. 1994a. Plastocyanin and the 33-kDa subimit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity. J. Biol. Chem. 269:18463-67. Yuan J, Henry R, McCafFery M, Cline K. 1994b. SecA homo log in protein transport within chloroplasts: evidence for endosymbiont-derived sorting. Science 266:796-98. Yuan J, Henry R, Cline K. 1993. Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70. Proc. Natl. Acad. Sci. USA 90:8552-56.

PAGE 99

94 Zak E, Sokolenko A, Unterholzner G, Altschmied L, Herrmann RG. 1997. On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes. Planta 201 :334-41 .

PAGE 100

BIOGRAPHICAL SKETCH Xianyue Ma was bom to a big femily on March 12, 1958, in Wuhan City, China. He completed high school in 1975. After working for three years in the fields on a farm, he continued his education at the Agriculture University of Central China where he received his bachelor's and master's degrees in crop sciences. In 1985, he was offered a position in Hubei University, where his main duty was teaching plant physiology to undergraduate students. In 1988, Hubei University sent him to England as a visiting researcher. He worked on a photoinhibition project in photosynthesis in Professor James Barber's laboratory for one year. He remained at Hubei University until 1992, when he came to United States to visit his wife. In order to gain laboratory experience in U.S., he worked for Dr. Daryl Pring at the University of Florida as an OPS. His jobs ranged from washing glassware to isolating mitochondria RNA. Xianyue Ma began his Ph.D. study in Dr. Kenneth Cline's laboratory in the fell of 1993, and he received his Ph. D. in May 1999. 95

PAGE 101

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fiilly adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. [ / ' ^ Kentfeth C. Cline, Chair Professor of Plant Molecular and Cellular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quahty, as a dissertation for the degree of Doctor of Philosophy. Alice Harmon Associate Professor of Plant Molecular and Cellular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fiilly adequate, in scope and quality, as a ^sertation for the degree of Doctor of Philosophy. 1} I fl Alfred S. Eewin Professor of Molecular Genetics I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Daryl R. Pring Professor of Plant Molecular and Cellular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fiilly adequate, in scope and quality, as a /lissertation for the degree of Doctor of Philosophy. Carlos Eduardo Vallejos Associate Professor of Plant Molecular and Cellular Biology

PAGE 102

This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirement for the degree of Doctor of Philosophy. May 1999 Dean, College o?Agriculture Dean, Graduate School


xml version 1.0 encoding UTF-8 standalone no
fcla fda yes
!-- Protein targeting and translocation on cpSec Delta pH pathways in chloroplast thylakoids ( Book ) --
METS:mets OBJID AA00030021_00001
xmlns:METS http:www.loc.govMETS
xmlns:xlink http:www.w3.org1999xlink
xmlns:xsi http:www.w3.org2001XMLSchema-instance
xmlns:daitss http:www.fcla.edudlsmddaitss
xmlns:mods http:www.loc.govmodsv3
xmlns:sobekcm http:digital.uflib.ufl.edumetadatasobekcm
xmlns:lom http:digital.uflib.ufl.edumetadatasobekcm_lom
xsi:schemaLocation
http:www.loc.govstandardsmetsmets.xsd
http:www.fcla.edudlsmddaitssdaitss.xsd
http:www.loc.govmodsv3mods-3-4.xsd
http:digital.uflib.ufl.edumetadatasobekcmsobekcm.xsd
METS:metsHdr CREATEDATE 2015-04-27T14:16:08Z ID LASTMODDATE 2015-03-13T10:02:34Z RECORDSTATUS COMPLETE
METS:agent ROLE CREATOR TYPE ORGANIZATION
METS:name UF,University of Florida
OTHERTYPE SOFTWARE OTHER
Go UFDC - FDA Preparation Tool
INDIVIDUAL
UFAD\renner
METS:dmdSec DMD1
METS:mdWrap MDTYPE MODS MIMETYPE textxml LABEL Metadata
METS:xmlData
mods:mods
mods:accessCondition The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
mods:genre authority marcgt bibliography
non-fiction
mods:identifier type ALEPH 030361454
OCLC 41876956
mods:language
mods:languageTerm text English
code iso639-2b eng
mods:location
mods:physicalLocation iuf
mods:name personal
mods:namePart Ma, Xianyue
given Xianyue
family Ma
date 1958-
mods:role
mods:roleTerm Main Entity
mods:note thesis Thesis (Ph.D.)--University of Florida, 1999.
bibliography Includes bibliographical references (leaves 85-94).
statement of responsibility by Xianyue Ma.
Typescript.
Vita.
mods:originInfo
mods:place
mods:placeTerm marccountry xx
mods:dateIssued marc 1999
point start 1999
mods:recordInfo
mods:recordIdentifier source sobekcm AA00030021_00001
mods:recordCreationDate 990706
mods:recordOrigin Imported from (ALEPH)030361454
mods:recordContentSource University of Florida
marcorg FUG
mods:languageOfCataloging
English
eng
mods:relatedItem original
mods:physicalDescription
mods:extent xi, 95 leaves : ill. ; 29 cm.
mods:subject SUBJ650_1 lcsh
mods:topic Plant proteins
Physiological transport
SUBJ650_2
Protein binding
SUBJ650_3
Thylakoids
SUBJ690_1
Plant Molecular and Cellular Biology thesis, Ph.D
SUBJ690_2
Dissertations, Academic
Plant Molecular and Cellular Biology
mods:geographic UF
mods:titleInfo
mods:title Protein targeting and translocation on cpSec and Delta pH pathways in chloroplast thylakoids
mods:typeOfResource text
DMD2
OTHERMDTYPE SOBEKCM SobekCM Custom
sobekcm:procParam
sobekcm:Aggregation ALL
UFIRG
UFIR
UFETD
IUF
VENDOR
VENDORIA
sobekcm:MainThumbnail proteintargeting00maxi_0079thm.jpg
sobekcm:Wordmark UFIR
UF
sobekcm:bibDesc
sobekcm:BibID AA00030021
sobekcm:VID 00001
sobekcm:EncodingLevel I
sobekcm:Source
sobekcm:statement UF University of Florida
METS:amdSec
METS:digiprovMD DIGIPROV1
DAITSS Archiving Information
daitss:daitss
daitss:AGREEMENT_INFO ACCOUNT PROJECT UFDC
METS:techMD TECH1
File Technical Details
sobekcm:FileInfo
sobekcm:File fileid JP21 width 2424 height 3289
JPEG1 630 855
JPEG2 853
JP22 3282
JPEG3
JP23 2420 3284
JPEG4
JP24 3283
JPEG5 856
JP25 3287
JPEG6
JP26 3285
JPEG7
JP27
JPEG8
JP28
JPEG9
JP29 2416
JPEG10
JP210
JPEG11 857
JP211
JPEG12 854
JP212 3276
JPEG13
JP213 3275
JPEG14
JP214
JPEG15
JP215
JPEG16
JP216 2412 3270
JPEG17
JP217 3273
JPEG18
JP218 3271
JPEG19
JP219 3272
JPEG20
JP220
JPEG21
JP221
JPEG22
JP222
JPEG23
JP223
JPEG24
JP224
JPEG25 859
JP225 2408
JPEG26 858
JP226 3279
JPEG27
JP227
JPEG28
JP228 2343 3181
JPEG29
JP229
JPEG30
JP230
JPEG31 860
JP231
JPEG32
JP232
JPEG33
JP233
JPEG34
JP234
JPEG35
JP235 2404 3268
JPEG36
JP236
JPEG37 863
JP237 3293
JPEG38
JP238
JPEG39
JP239
JPEG40
JP240 3269
JPEG41
JP241
JPEG42 862
JP242 3288
JPEG43
JP243
JPEG44
JP244
JPEG45
JP245
JPEG46
JP246 2400
JPEG47
JP247
JPEG48
JP248
JPEG49
JP249
JPEG50
JP250
JPEG51
JP251
JPEG52 861
JP252 3281
JPEG53
JP253
JPEG54
JP254
JPEG55
JP255
JPEG56
JP256 2396 3280
JPEG57
JP257
JPEG58
JP258
JPEG59
JP259 2380 3235
JPEG60
JP260 3265
JPEG61
JP261 3263
JPEG62
JP262
JPEG63 864
JP263 2392
JPEG64
JP264
JPEG65
JP265
JPEG66 866
JP266
JPEG67
JP267
JPEG68
JP268
JPEG69
JP269
JPEG70
JP270
JPEG71
JP271
JPEG72
JP272
JPEG73
JP273
JPEG74 865
JP274
JPEG75
JP275 2388
JPEG76
JP276
JPEG77
JP277
JPEG78 868
JP278
JPEG79
JP279
JPEG80
JP280
JPEG81
JP281
JPEG82
JP282
JPEG83
JP283
JPEG84
JP284
JPEG85
JP285 2384
JPEG86 867
JP286
JPEG87 869
JP287
JPEG88
JP288
JPEG89
JP289
JPEG90
JP290
JPEG91
JP291
JPEG92
JP292
JPEG93 871
JP293
JPEG94
JP294
JPEG95
JP295
JPEG96
JP296
JPEG97
JP297
JPEG98
JP298
JPEG99
JP299
JPEG100
JP2100 2376
JPEG101 878
JP2101 2348
JPEG102 876
JP2102 2352
JPEG103 877
JP2103
METS:fileSec
METS:fileGrp USE archive
METS:file GROUPID G1 TIF1 imagetiff CHECKSUM 41c03cfc3584b01f27770d4d5f360e70 CHECKSUMTYPE MD5 SIZE 23930388
METS:FLocat LOCTYPE OTHERLOCTYPE SYSTEM xlink:href proteintargeting00maxi_0000.tif
G2 TIF2 ecfcb28084ddc74eff2762a43d3014da 23880792
proteintargeting00maxi_0001.tif
G3 TIF3 936e2520d38c968c4b527943a1eafce5 23855900
proteintargeting00maxi_0002.tif
G4 TIF4 5665659f793be0e13a1d214b1d835732 23847476
proteintargeting00maxi_0003.tif
G5 TIF5 39501116ff253271714474acd0aa5ff9 23878572
proteintargeting00maxi_0004.tif
G6 TIF6 2a5507a67318258b13f9bc46ba5db84a 23863976
proteintargeting00maxi_0005.tif
G7 TIF7 536652796a6801156e571fa8e76c13cb 23856756
proteintargeting00maxi_0006.tif
G8 TIF8 c6ce79c15475c40424d00459cfea05b5 23850092
proteintargeting00maxi_0007.tif
G9 TIF9 6713ecca37246db21e313b76d8cef8fc 23810520
proteintargeting00maxi_0008.tif
G10 TIF10 82e4a6c6a187471fc81b46cfeb557cbd 23818516
proteintargeting00maxi_0009.tif
G11 TIF11 3772b7390d6be08bd525214c06962db5 23825280
proteintargeting00maxi_0010.tif
G12 TIF12 dbc35556c1c263a859bf3f51836228eb 23760240
proteintargeting00maxi_0011.tif
G13 TIF13 7416f5cfe09b2ad8ebd3876e0ec2ad11 23753076
proteintargeting00maxi_0012.tif
G14 TIF14 b33402182d452853aea76f008264dc05 23823912
proteintargeting00maxi_0013.tif
G15 TIF15 64d6f9691984ced49e8bbd92a86deed4 23825820
proteintargeting00maxi_0014.tif
G16 TIF16 1e893f07945eb5bb2223794dd0a54363 23676156
proteintargeting00maxi_0015.tif
G17 TIF17 0bbc125d092416a0066677e4edc90701 23738292
proteintargeting00maxi_0016.tif
G18 TIF18 3d26f6fd4733ef7db5ea69ecf8773a53 23684776
proteintargeting00maxi_0017.tif
G19 TIF19 9e16bcd5a3a12b745ffd63314724285f 23691844
proteintargeting00maxi_0018.tif
G20 TIF20 da6609f259bf05e5d61d181f6748e19d 23713668
proteintargeting00maxi_0019.tif
G21 TIF21 3c221a9d92f0d7fa07ae343569c6bf83 23771328
proteintargeting00maxi_0020.tif
G22 TIF22 d0826d925db577eb1f674801f172d0ed 23771896
proteintargeting00maxi_0021.tif
G23 TIF23 14cf9553a08639b516aaf3fd30c68e12 23721204
proteintargeting00maxi_0022.tif
G24 TIF24 324e143a1603a5ddd7555944ce9b9603 23771404
proteintargeting00maxi_0023.tif
G25 TIF25 b70c4b59dd45d9d025a4a58b6c485ed2 23724820
proteintargeting00maxi_0024.tif
G26 TIF26 8c962bca3309f24481ad7f76bf3f00e7 23702984
proteintargeting00maxi_0025.tif
G27 TIF27 e44419475310fce52211337ebeaffff8 23722096
proteintargeting00maxi_0026.tif
G28 TIF28 7c8507a1c57634d609344399e6190539 22383420
proteintargeting00maxi_0056a.tif
G29 TIF29 e413e427115d2c92e112934df848f152 23703216
proteintargeting00maxi_0027.tif
G30 TIF30 c3b93f4f0ecd07923c78d20800a6384b 23746728
proteintargeting00maxi_0028.tif
G31 TIF31 fba459ac2ed3e531e2bb64fbcd7a37c8 23776056
proteintargeting00maxi_0029.tif
G32 TIF32 dc8f96755ef8ecc418c00b15bcf5548a 23702932
proteintargeting00maxi_0030.tif
G33 TIF33 4d06bce64ef2054135e11241bf64ab0b 23725572
proteintargeting00maxi_0031.tif
G34 TIF34 1328d23509cede380632a4eba38f902e 23703608
proteintargeting00maxi_0032.tif
G35 TIF35 4b4e34c932597de3282e23b19ddff24e 23585100
proteintargeting00maxi_0033.tif
G36 TIF36 42dfa24aa9f68dcdf19d6d36284bb566 23692428
proteintargeting00maxi_0034.tif
G37 TIF37 372922efe3df8cc24f1c3081c2f48517 23765092
proteintargeting00maxi_0035.tif
G38 TIF38 a9c331a5aa459496bb0f7305a13c9a1c 23597568
proteintargeting00maxi_0036.tif
G39 TIF39 6df89ea85f433b0274753bc2b6a6adb6 23685712
proteintargeting00maxi_0037.tif
G40 TIF40 4259d93c5f51e4bc1ba38b7aeb89dccc 23591408
proteintargeting00maxi_0038.tif
G41 TIF41 ca0d391edd97ebc690d08d0004cf7474 23603600
proteintargeting00maxi_0039.tif
G42 TIF42 108cdb9b569c7eb5f0ebc5f19d286912 23729224
proteintargeting00maxi_0040.tif
G43 TIF43 27edcf17dc4efebf11531a0ffa045f2e 23734812
proteintargeting00maxi_0041.tif
G44 TIF44 cb90fdf6c4282e06f8259045dfefffd3 23765036
proteintargeting00maxi_0042.tif
G45 TIF45 b2ee34ec96f7bf8658e5f43fc2c90e68 23734952
proteintargeting00maxi_0043.tif
G46 TIF46 db8dd47f72bb779c546f084eea4e4ab8 23646608
proteintargeting00maxi_0044.tif
G47 TIF47 b93c100de2346acaf0b506fedbf4c60e 23696224
proteintargeting00maxi_0045.tif
G48 TIF48 5a14a166d133fe81a041ea65de49a025 23660492
proteintargeting00maxi_0046.tif
G49 TIF49 ac53fa997501b73455f51cb5ccd23edf 23695556
proteintargeting00maxi_0047.tif
G50 TIF50 f632f1a0b6d89b66607e648723bed1dd 23646364
proteintargeting00maxi_0048.tif
G51 TIF51 2273f5365bb37004b5257a42bb22c04e 23694092
proteintargeting00maxi_0049.tif
G52 TIF52 b09fecfd543aa13fc56d8cd2f5a54489 23638068
proteintargeting00maxi_0050.tif
G53 TIF53 b5a62b144b0cc1ee0bc57e81bd8fcb89 23653312
proteintargeting00maxi_0051.tif
G54 TIF54 5eca6a11f5b6e28bc7beaea3192b977a 23653840
proteintargeting00maxi_0052.tif
G55 TIF55 3c62e58d35a5187b7a14022b2f74f393 23574648
proteintargeting00maxi_0053.tif
G56 TIF56 7f85b99c424297c3eb33228ec581ea9a 23592856
proteintargeting00maxi_0054.tif
G57 TIF57 5ab72faded949e2954ade6a5fcd15bf1 23535260
proteintargeting00maxi_0055.tif
G58 TIF58 773d903a575cb2fa681f191c883ce910 23528188
proteintargeting00maxi_0056.tif
G59 TIF59 f7b7f41a0dd40cd972b5b1538fd80e4c 23110564
proteintargeting00maxi_0057.tif
G60 TIF60 329ca9ff48502fa28841161a3efc9366 23484244
proteintargeting00maxi_0058.tif
G61 TIF61 fbfb01ca41cf9f41adad11b2ce7227ff 23470300
proteintargeting00maxi_0059.tif
G62 TIF62 b26794300723d5b4fdf21ef0bb891538 23556120
proteintargeting00maxi_0060.tif
G63 TIF63 375ae3bcef318382c8005769ec182385 23546112
proteintargeting00maxi_0061.tif
G64 TIF64 bd843294a91ef3707c80e372f549ba79 23585484
proteintargeting00maxi_0062.tif
G65 TIF65 cc8ed4aa15ac4617e69a148a23222d29 23545696
proteintargeting00maxi_0063.tif
G66 TIF66 8be78168ed9912b18f05915a3e9f26d2 23617896
proteintargeting00maxi_0064.tif
G67 TIF67 69f71807339f458d42cbfb1e09a63259 23544508
proteintargeting00maxi_0065.tif
G68 TIF68 3523d13316bb883c4b7e483b242641a3 23618024
proteintargeting00maxi_0066.tif
G69 TIF69 f807750db43eb1141f34dfe97cefe7d7 23545704
proteintargeting00maxi_0067.tif
G70 TIF70 07927f9e7a24c80e4b5f31be8bdcfdc3 23546188
proteintargeting00maxi_0068.tif
G71 TIF71 39ad547886dd44d7d301ffebc8eedde6 23617824
proteintargeting00maxi_0069.tif
G72 TIF72 a39e0dd220fa30bc3e168e6326c9d0d0 23544304
proteintargeting00maxi_0070.tif
G73 TIF73 363c4f76320cd7373988754195b8d4e6 23616884
proteintargeting00maxi_0071.tif
G74 TIF74 52b101048b342e3e87baacc287433ec5 23574528
proteintargeting00maxi_0072.tif
G75 TIF75 a2579ff5a9a2aea1b92bcca9743e5bbc 23505600
proteintargeting00maxi_0073.tif
G76 TIF76 4866c2a71248079f4b225e6b46e72a75 23506560
proteintargeting00maxi_0074.tif
G77 TIF77 f890532466cf24f9879dc7ce47dd5b53 23505568
proteintargeting00maxi_0075.tif
G78 TIF78 78b397081395335fb2ecca51198abcea 23577456
proteintargeting00maxi_0076.tif
G79 TIF79 4e3f5e62f4b79b15824de18f154a7c2d 23506876
proteintargeting00maxi_0077.tif
G80 TIF80 07957180c886d6eee4e51fbbcf2450ca 23578212
proteintargeting00maxi_0078.tif
G81 TIF81 d4b4515c7286d6f1d4e767b93d3c875e 23505488
proteintargeting00maxi_0079.tif
G82 TIF82 56396c44f65b214b720ff1a3ba7a16ba 23504556
proteintargeting00maxi_0080.tif
G83 TIF83 254703b79acc2d4b6270a1937fbb743b 23542536
proteintargeting00maxi_0081.tif
G84 TIF84 8ad2d9207dfd0bc9a179de1e28efa1cb 23505232
proteintargeting00maxi_0082.tif
G85 TIF85 a3aa753ad9be3a92ad2d507b2db637fc 23445896
proteintargeting00maxi_0083.tif
G86 TIF86 569c9d14095df1ed09f0f16e4d254378 23467844
proteintargeting00maxi_0084.tif
G87 TIF87 88c0612c9f5c5afefc2c732a0fd99bb1 23539328
proteintargeting00maxi_0085.tif
G88 TIF88 1034439be35779c4240404a27783c96c 23502048
proteintargeting00maxi_0086.tif
G89 TIF89 127b8cf181f3571ad3f69ca409eeb092 23402536
proteintargeting00maxi_0087.tif
G90 TIF90 655cbf51e26f4a54475d4f4b8061c300 23510112
proteintargeting00maxi_0088.tif
G91 TIF91 d251210d1a076c36e82a9009a60bf7bc 23503196
proteintargeting00maxi_0089.tif
G92 TIF92 4e5c5cc94197b85eca0453a7f09efc4e 23503988
proteintargeting00maxi_0090.tif
G93 TIF93 4b864868796509fe6499ac17e1b67197 23499988
proteintargeting00maxi_0091.tif
G94 TIF94 d8bf088408739502a797fe36d7d6ad94 23450404
proteintargeting00maxi_0092.tif
G95 TIF95 3e9bbc9464c703101dd31def39e9855d 23500124
proteintargeting00maxi_0093.tif
G96 TIF96 95a1bf9c6d3c56979d1cdda30c831810 23457264
proteintargeting00maxi_0094.tif
G97 TIF97 bf471aad950a301a58615c292e79e478 23400336
proteintargeting00maxi_0095.tif
G98 TIF98 93f549856fe6b163e5d2d188469f2830 23450252
proteintargeting00maxi_0096.tif
G99 TIF99 5139d33b3acda80eeb268844e9ac6c43 23436048
proteintargeting00maxi_0097.tif
G100 TIF100 837f17dcb871d2916f294a6966768911 23335096
proteintargeting00maxi_0098.tif
G101 TIF101 38a37969b44be05288964ea5cbbcf02d 23062492
proteintargeting00maxi_0099.tif
G102 TIF102 fa504cfbf7efbd131ea9904823a51e67 23102120
proteintargeting00maxi_0100.tif
G103 TIF103 dfeb6d906e379c6d68877c64151eeb89 23120980
proteintargeting00maxi_0101.tif
reference
imagejp2 a82a5e9538c88e93dd775e2068f2232e 233787
proteintargeting00maxi_0000.jp2
c2ce49258e288ecd7a85e597710b5812 392323
proteintargeting00maxi_0001.jp2
9e5527e59ad4e234726467de1437d8f0 497496
proteintargeting00maxi_0002.jp2
99523fa6ff5fe207c88261cb3a6d77a4 278791
proteintargeting00maxi_0003.jp2
263d80b34a00d0793ec2858fc9b79a42 529233
proteintargeting00maxi_0004.jp2
cc4b0f12bf4c3de0166add3a1bea8a19 507788
proteintargeting00maxi_0005.jp2
632c12d5362af5e1607e88e5fbd01d33 519851
proteintargeting00maxi_0006.jp2
9c6ca70bcba38e2257d9981fadad8b73 589673
proteintargeting00maxi_0007.jp2
9282e3b71fcae68993d5ed2749c851f4 568924
proteintargeting00maxi_0008.jp2
c556542957ce94300b38f10b75f8e055 677456
proteintargeting00maxi_0009.jp2
f091377d250f7355dc894626683a423e 589339
proteintargeting00maxi_0010.jp2
81d95531ffdeef7dffad657b844a6dde 623243
proteintargeting00maxi_0011.jp2
077fed465d6d51911dd03b76683a140e 631905
proteintargeting00maxi_0012.jp2
6ff555622cbdacde0068bba541c2fe50 544624
proteintargeting00maxi_0013.jp2
c870b88c7c7c035aaf9b151adff15e46 641965
proteintargeting00maxi_0014.jp2
256f652e54611e987eba06ef33131104 467196
proteintargeting00maxi_0015.jp2
5b1f38a00ccd6f00009f3577fc121dc9 611319
proteintargeting00maxi_0016.jp2
90182db99a433068533d55cb8257caa5 636019
proteintargeting00maxi_0017.jp2
69672c4d98f0be6ced17496b83131393 596045
proteintargeting00maxi_0018.jp2
e9fcbfa521748b24f393f7b9c89ede16 627115
proteintargeting00maxi_0019.jp2
208a7b16f602791dc7338105c70361bb 592703
proteintargeting00maxi_0020.jp2
c980ae149de17379c1978b3dc0bc1f4e 647734
proteintargeting00maxi_0021.jp2
1885f193e6e5e32e61b85d6a7c9188ab 640733
proteintargeting00maxi_0022.jp2
53f88a069b61c4eef289b9237b942f65 565358
proteintargeting00maxi_0023.jp2
9759bb53bc47437a5497780d137d06e0 580388
proteintargeting00maxi_0024.jp2
caa9a621ada6f26e7ac6b44ae0013f7a 647521
proteintargeting00maxi_0025.jp2
3927c790fb28a7dfea6570139d3fee05 217158
proteintargeting00maxi_0026.jp2
2e9622b7b8da7da533dd32ebfa6bb7ab 931722
proteintargeting00maxi_0056a.jp2
0209f537038dd39a2a7e0ae58721f9dc 610673
proteintargeting00maxi_0027.jp2
5023692e2a72ffc3c75d4bde51583059 621310
proteintargeting00maxi_0028.jp2
401e6a91679389cc62f4af6353de04f4 648176
proteintargeting00maxi_0029.jp2
6ba6a545eb6321faadd70922ae98bc3f 578594
proteintargeting00maxi_0030.jp2
276cc46a853e17c6d46423860dbfd600 645851
proteintargeting00maxi_0031.jp2
57cb1dc8668b50f21921d730a56d6cf2 683219
proteintargeting00maxi_0032.jp2
edd6e902675c9e907877baf5f014b4fd 617286
proteintargeting00maxi_0033.jp2
03680c79412a588a854a306b04e42400 584062
proteintargeting00maxi_0034.jp2
2eaa028d537428722c98e5501fe6c161 630712
proteintargeting00maxi_0035.jp2
644234d914a7e536b626f35dce8a25e5 466266
proteintargeting00maxi_0036.jp2
b0fb480a615a7972afcd91809f68ac3d 611567
proteintargeting00maxi_0037.jp2
8bb0cf8b4bb47075fb0e8e4ff267624c 611962
proteintargeting00maxi_0038.jp2
ae502d30dea2f4584efb607827741910 261186
proteintargeting00maxi_0039.jp2
002acd3c235f57515f4eb5e325bf3be7 649396
proteintargeting00maxi_0040.jp2
5806c69289552464c5a60656b4a7a758 519129
proteintargeting00maxi_0041.jp2
2aef7afe49df6525291b86f41baa9d69 612443
proteintargeting00maxi_0042.jp2
8176be89fbb360c7c137665a3c8b93a5 525755
proteintargeting00maxi_0043.jp2
0266fcd00849cfb5cdbd2d71b17ee545 653026
proteintargeting00maxi_0044.jp2
32d869ecfb87987465c7919d56f53c00 683998
proteintargeting00maxi_0045.jp2
a1fab421bb31907915be719651b05bcf 616920
proteintargeting00maxi_0046.jp2
08b0c5ed3890a7d3a3fbe5c5fcdb112b 585902
proteintargeting00maxi_0047.jp2
fa6bcccdf542a5ab24afbc29df9cae11 634211
proteintargeting00maxi_0048.jp2
a383c52527ab90c30474f3f6a3b52838 258487
proteintargeting00maxi_0049.jp2
08dacf29b7c74f9d3e971dfc110ca424 571784
proteintargeting00maxi_0050.jp2
2ffa43851cf1cdad8a757836b3b64b8a 608558
proteintargeting00maxi_0051.jp2
21e39088da5c43a204e0a71345879307 669499
proteintargeting00maxi_0052.jp2
d62edf4712fa254364ed89aecba41bb3 654340
proteintargeting00maxi_0053.jp2
6134e3f85a95394577b5fadb241107b4 675545
proteintargeting00maxi_0054.jp2
e0053922623c41e831c773a956cd7e8f 658356
proteintargeting00maxi_0055.jp2
3705b056c7f33988335e591c224451ee 654334
proteintargeting00maxi_0056.jp2
b091dfe8f9504f42b60a372fcee391cc 161549
proteintargeting00maxi_0057.jp2
ed87b8776d8d9913891c1b522644bbe0 545551
proteintargeting00maxi_0058.jp2
a4740d70c88e722a34c571de27557637 612724
proteintargeting00maxi_0059.jp2
fbb034b99a703beede345e4d312d85af 573730
proteintargeting00maxi_0060.jp2
5a22ad40d3655410e78b523c18f8db42 690576
proteintargeting00maxi_0061.jp2
453a0609ede722c4592a028ffa057915 658030
proteintargeting00maxi_0062.jp2
33950eceec6e9d75340dfccb1d9ae5b2 585925
proteintargeting00maxi_0063.jp2
6e8028ef8b9bd63c3ea12cb30dff7bbc 658551
proteintargeting00maxi_0064.jp2
7cadeaeff112e8333048bf5dbd050989 571905
proteintargeting00maxi_0065.jp2
8d21ed4d6e96d009c24aa28f1ad6175d 626045
proteintargeting00maxi_0066.jp2
7123015d5e77c22ae56e846335acd007 727114
proteintargeting00maxi_0067.jp2
acdd8fe13d5355453008a87dac1699b9 700607
proteintargeting00maxi_0068.jp2
30d139fea3bd78776a65c853b6244000 596412
proteintargeting00maxi_0069.jp2
d99210b16203535282999adb0739aac8 511557
proteintargeting00maxi_0070.jp2
19e83d0c391a885fec28f883b638450d 621145
proteintargeting00maxi_0071.jp2
b9c110f937f346dfd6822bd330a5380d 654541
proteintargeting00maxi_0072.jp2
75f6bb30d89682b2bfc5184a2fa7f686 681014
proteintargeting00maxi_0073.jp2
3b802d23d9d06369c8e37bde392a15ab 655694
proteintargeting00maxi_0074.jp2
3004853f8f557b70297847ce1f8f0d08 664282
proteintargeting00maxi_0075.jp2
1262ea5328670bf6c08384dfddced5e9 530527
proteintargeting00maxi_0076.jp2
a10c380334b0c4d87f7b032c979c4d5a 698891
proteintargeting00maxi_0077.jp2
a271f90a9b47355e8468d4984e9a99ad 583047
proteintargeting00maxi_0078.jp2
592246232d214cb98b4f98bd76525b89 619676
proteintargeting00maxi_0079.jp2
70f0aac71c8303176c2aac3034da5f97 373097
proteintargeting00maxi_0080.jp2
06461e6395720aa612a5087ee0db2401 612935
proteintargeting00maxi_0081.jp2
e00c59fe735c036401e054eb859a6dd0 507582
proteintargeting00maxi_0082.jp2
6a0d0c394047820bda9b016540fe2c41 630208
proteintargeting00maxi_0083.jp2
e6b0e2e9eb4b6c0fe3dd284924248f87 745237
proteintargeting00maxi_0084.jp2
ce6a2ac69ec436019ada3541e31b6ce9 685371
proteintargeting00maxi_0085.jp2
ac414bfbe623ac3554701c2621eb032d 493804
proteintargeting00maxi_0086.jp2
53f75c919e2fe24542b9998e870d4d06 546000
proteintargeting00maxi_0087.jp2
86fd040daebc019a753e5e55b141e543 599702
proteintargeting00maxi_0088.jp2
2e743e6490cdbb70f81a7208c1db9307 684313
proteintargeting00maxi_0089.jp2
b38f7197927425500acf0b663ad95827 764999
proteintargeting00maxi_0090.jp2
76e80a6b0f618e37e8138d07bc56fef7 763341
proteintargeting00maxi_0091.jp2
c7aa5b8e7ee54b07665ac2383cb52c55 781490
proteintargeting00maxi_0092.jp2
a5e6c28d8d01ba9b41d1b07c04e0ec74 751104
proteintargeting00maxi_0093.jp2
0fefd635a9869d8af6e781af8fb56773 755548
proteintargeting00maxi_0094.jp2
023afb2d74d821f073572eb93639fb9a 744517
proteintargeting00maxi_0095.jp2
34098b1ab995cb558f2f0f51594ccd88 765301
proteintargeting00maxi_0096.jp2
57fea7f1b77a2d7010aee453bf40b3e2 778145
proteintargeting00maxi_0097.jp2
727b8df1106f54bb9c170a05133fc238 138856
proteintargeting00maxi_0098.jp2
d659ac15a06a60c9ddba29cc17667d18 431229
proteintargeting00maxi_0099.jp2
36763cabc2fa1ceb7b7157f1af303d75 542583
proteintargeting00maxi_0100.jp2
da00b8216d238f78e3f4b39cf45b077c 141377
proteintargeting00maxi_0101.jp2
imagejpeg d27be7686c7ac9fb84de6e4c5e4f886b 55705
proteintargeting00maxi_0000.jpg
JPEG1.2 2a601c2e86a6c01735dc8285f84100f1 25704
proteintargeting00maxi_0000.QC.jpg
67c7d294565f32279a5a6580767ce52b 88404
proteintargeting00maxi_0001.jpg
JPEG2.2 115e261c034580c784f4cebcd1b9d368 37147
proteintargeting00maxi_0001.QC.jpg
8a226e9287c1bbf37a96bc1b86fae560 105263
proteintargeting00maxi_0002.jpg
JPEG3.2 465d904ecf963e03db58745feffabcb6 39078
proteintargeting00maxi_0002.QC.jpg
bc045f26dabb3aed8bc5af4171d47a3d 63204
proteintargeting00maxi_0003.jpg
JPEG4.2 2f38355c3c4c2b63e9f3e140206695ac 26868
proteintargeting00maxi_0003.QC.jpg
a70ba38ff8d86f77086224f4101f354a 116438
proteintargeting00maxi_0004.jpg
JPEG5.2 f8c7b236a455ae3e8794510111a80e27 46784
proteintargeting00maxi_0004.QC.jpg
574ef92cb64e7a3ea37070c36ad4671a 107736
proteintargeting00maxi_0005.jpg
JPEG6.2 dc1f7569aeb1df9e39ada88bc6226cc1 43170
proteintargeting00maxi_0005.QC.jpg
c3f0fba2c9380f99e3c4e708e9e9b048 110309
proteintargeting00maxi_0006.jpg
JPEG7.2 e7904821b3233398fb587bc478efb3d4 45170
proteintargeting00maxi_0006.QC.jpg
5e869eac112279a524f9ff5c12d9b102 125240
proteintargeting00maxi_0007.jpg
JPEG8.2 ef65e4536935b6ba372028f3d415b49f 51479
proteintargeting00maxi_0007.QC.jpg
1972f37e828c509e254f49400e303ed9 116319
proteintargeting00maxi_0008.jpg
JPEG9.2 0ed3b7ed1183b6166697c62102679e36 49304
proteintargeting00maxi_0008.QC.jpg
9b71ba18aaa6d3d45ad05dcd8cf75564 142886
proteintargeting00maxi_0009.jpg
JPEG10.2 90d8f1314ba43757d9a34ba128da3121 57730
proteintargeting00maxi_0009.QC.jpg
1ba601bf6bd757552f1d97ee825e439c 124267
proteintargeting00maxi_0010.jpg
JPEG11.2 07488b5c4dae4f045e928620b4cc3c58 52713
proteintargeting00maxi_0010.QC.jpg
255aded46931b87e75a8500847f1993c 130927
proteintargeting00maxi_0011.jpg
JPEG12.2 ddd4f19fd9f829296f2baedf276fd3bf 54715
proteintargeting00maxi_0011.QC.jpg
8c07c196b822ef0e0f3c4561d9cde835 133992
proteintargeting00maxi_0012.jpg
JPEG13.2 96dbde60fb51d0a3c82e0e85c8a78ba2 54277
proteintargeting00maxi_0012.QC.jpg
d80be01c51beb5a86389c8edae9ead5b 83738
proteintargeting00maxi_0013.jpg
JPEG14.2 29cd3f554463762f2c2504439f862d9d 34979
proteintargeting00maxi_0013.QC.jpg
b8c93baf2363dac9926ff8ebd1c6d7fd 140311
proteintargeting00maxi_0014.jpg
JPEG15.2 be4590b328e7b336af2cb9b36736f9ce 57328
proteintargeting00maxi_0014.QC.jpg
40f4a4ce3fd9e8b7bf61ae47a5f938d3 102948
proteintargeting00maxi_0015.jpg
JPEG16.2 0e5628415efed7f28e5299b157ef095a 40498
proteintargeting00maxi_0015.QC.jpg
0f2c43ae8495e66a84cbcd2891c7c7e2 127960
proteintargeting00maxi_0016.jpg
JPEG17.2 edaaf300276dc21c224bdf826804c9e4 51781
proteintargeting00maxi_0016.QC.jpg
1ecc4bc37f66b135a0ec521fa3b11fa2 134467
proteintargeting00maxi_0017.jpg
JPEG18.2 24abf5e56456dd5db8decad185b0cfef 54127
proteintargeting00maxi_0017.QC.jpg
b0a630a39ac7a8f1755eb253ad9f1fcf 124547
proteintargeting00maxi_0018.jpg
JPEG19.2 284649e07cfe3f483eacecb252ea566d 52933
proteintargeting00maxi_0018.QC.jpg
88788fa4f2ac611b73b82216f8fb5497 132601
proteintargeting00maxi_0019.jpg
JPEG20.2 746af1a51347393893e176b54af07b89 53547
proteintargeting00maxi_0019.QC.jpg
89fd75144b6bef39453a3041f9fe01f9 127886
proteintargeting00maxi_0020.jpg
JPEG21.2 8587c0d70757bf4caf0393aa93d358bb 52694
proteintargeting00maxi_0020.QC.jpg
75f0eb2ba25f438941a9ca099cb5157e 139618
proteintargeting00maxi_0021.jpg
JPEG22.2 e2a35f5713e7449d751e96b132aa4784 57606
proteintargeting00maxi_0021.QC.jpg
bd459192ce2d4496144f2ec3d05a65e4 136879
proteintargeting00maxi_0022.jpg
JPEG23.2 83d59eab9b286cec9794003c16cd9efa 56345
proteintargeting00maxi_0022.QC.jpg
7e5045182f26cb8811427870cfda60c8 119435
proteintargeting00maxi_0023.jpg
JPEG24.2 563b272a1496adbb8b5b1a4de72a4939 49867
proteintargeting00maxi_0023.QC.jpg
81bf3b4378f6656795c757c2fa6ba29e 126663
proteintargeting00maxi_0024.jpg
JPEG25.2 bdec3229fa84c63951dd8d7240fc9d84 53436
proteintargeting00maxi_0024.QC.jpg
b09c3f2e5731a10262d55217f5081fe2 127763
proteintargeting00maxi_0025.jpg
JPEG26.2 37b61a2fb3582387215de0a993e65843 52732
proteintargeting00maxi_0025.QC.jpg
553d77980eb4e4117d059a02cc7a15ad 53706
proteintargeting00maxi_0026.jpg
JPEG27.2 119e92f06dd4dac456e00f223178f958 27047
proteintargeting00maxi_0026.QC.jpg
2c361b9f43c49515fa7e7674ddf2a672
proteintargeting00maxi_0056a.jpg
JPEG28.2 a7b5b55d04810dd2923720bfdbb52f1e 64527
proteintargeting00maxi_0056a.QC.jpg
e3ffb9aa3bb2e23f563675131e3162c6 126986
proteintargeting00maxi_0027.jpg
JPEG29.2 599b55261ed4f43e0553444d0b6ee54b 52207
proteintargeting00maxi_0027.QC.jpg
532f790f83ad4694982e8401c592dd33 130342
proteintargeting00maxi_0028.jpg
JPEG30.2 9681607904df7fe0ef15d81d4ecfd65b 55169
proteintargeting00maxi_0028.QC.jpg
839552ad2cc93a57a0839925bc8af9a2 134398
proteintargeting00maxi_0029.jpg
JPEG31.2 80d60e16a9f743506bd56dc572649abb 57362
proteintargeting00maxi_0029.QC.jpg
fb07b60fff129298b2be7844eb3e3737 114562
proteintargeting00maxi_0030.jpg
JPEG32.2 834ea7146699bc4dc61b441e8499033a 49680
proteintargeting00maxi_0030.QC.jpg
54aca7f518c7add599317145465fefa7 143993
proteintargeting00maxi_0031.jpg
JPEG33.2 07bb27ad136bea2df846a7c0cf164508 60448
proteintargeting00maxi_0031.QC.jpg
ac29a706c615b43abbb9d5b4999db1d2 139034
proteintargeting00maxi_0032.jpg
JPEG34.2 eae50c3c305d88c120a6024f9a96169e 58854
proteintargeting00maxi_0032.QC.jpg
be7358aed8559c92a4e7d8d51b4583a6 131186
proteintargeting00maxi_0033.jpg
JPEG35.2 d82ac3324971f971e401695a7808c07d 56168
proteintargeting00maxi_0033.QC.jpg
5f8cd73c5f32f6d76b7e5ba1e060e147 121488
proteintargeting00maxi_0034.jpg
JPEG36.2 3f9d106bb5cf1d6656ab5a851d61f062 50514
proteintargeting00maxi_0034.QC.jpg
5177d14d8586cbbd481d693b3c6f46a8 132714
proteintargeting00maxi_0035.jpg
JPEG37.2 c06744b2c03b6d73caf8c43e717eea28 55654
proteintargeting00maxi_0035.QC.jpg
e8e6053e9727e403bea8e7236c83df41 101529
proteintargeting00maxi_0036.jpg
JPEG38.2 0e8be429880aea697dc494cd94595db3 39761
proteintargeting00maxi_0036.QC.jpg
4218e5b8a0983f9d0a82f9ddb7219f46 128674
proteintargeting00maxi_0037.jpg
JPEG39.2 0f366bc18d50d0e5dbdaa6f77cadf983 52832
proteintargeting00maxi_0037.QC.jpg
71a1c8cfee44d89cf51166cea613dce0 135330
proteintargeting00maxi_0038.jpg
JPEG40.2 484b9eb751fe7dc5205a557bd1d38e2e 48254
proteintargeting00maxi_0038.QC.jpg
e9aae4fa5d47953441c184a9f314d367 60958
proteintargeting00maxi_0039.jpg
JPEG41.2 470321573ac55bfdb4fc22e90d3c8012 27617
proteintargeting00maxi_0039.QC.jpg
80bcb547ef2c5c64fe21e82226436f4a 137477
proteintargeting00maxi_0040.jpg
JPEG42.2 1f42f5d05d2f1d7445a5b64dfe6ee555 55291
proteintargeting00maxi_0040.QC.jpg
1791bcb4ef2b0f224f2982eb8d7c284a 104612
proteintargeting00maxi_0041.jpg
JPEG43.2 8c613a43482a02af65406c69375fa356 41185
proteintargeting00maxi_0041.QC.jpg
29ce702785b41f70b57d2d92e8a0f3ae 131346
proteintargeting00maxi_0042.jpg
JPEG44.2 71f017742a0717058c760e0e703835ec 54550
proteintargeting00maxi_0042.QC.jpg
c81341e0ca02d5bf658e045bf58112b6 109960
proteintargeting00maxi_0043.jpg
JPEG45.2 64706a13027be443cc0c92b7c7d2f3ba 42127
proteintargeting00maxi_0043.QC.jpg
6a586259320d70724914351a79bd353a 136547
proteintargeting00maxi_0044.jpg
JPEG46.2 24152c05b2c044b4e6bcc6a9caa7704e 56858
proteintargeting00maxi_0044.QC.jpg
e7aaa9fc4e06ceb4123121f4d6b7f16f 134922
proteintargeting00maxi_0045.jpg
JPEG47.2 9e503a7fea2cdaa46c9e5bdcd8ec23de 49716
proteintargeting00maxi_0045.QC.jpg
4d7aef000bea9838730563b3b6f16d7f 129994
proteintargeting00maxi_0046.jpg
JPEG48.2 1d513b7a8d5316b7078417f02ba38736 54214
proteintargeting00maxi_0046.QC.jpg
44c7ca3b8d5a1e8ae308a15da1fa7788 124287
proteintargeting00maxi_0047.jpg
JPEG49.2 778f19b54c206a5b93b163fd7e1359b4 44434
proteintargeting00maxi_0047.QC.jpg
a51197dae2062a9727e4f30ba1e940b9 132635
proteintargeting00maxi_0048.jpg
JPEG50.2 4a3720c81bdf2ba3b93fbeff8e12ecc9 55737
proteintargeting00maxi_0048.QC.jpg
6b5afebb841b8fef127ba45e908cc3c0 64278
proteintargeting00maxi_0049.jpg
JPEG51.2 951cdcc51aebf130427db5a73bd324bc 27642
proteintargeting00maxi_0049.QC.jpg
90602f7cc36a9e3f39b7c96ac2b17a41 123034
proteintargeting00maxi_0050.jpg
JPEG52.2 776b7d0a4b51e099daa09c675f1ac4c4 43975
proteintargeting00maxi_0050.QC.jpg
841a698545c83873373585a4ed73556b 127117
proteintargeting00maxi_0051.jpg
JPEG53.2 e38d3f6477e514ecf3a6d9b873f40940 53625
proteintargeting00maxi_0051.QC.jpg
6c7727f1f9b57d1cd20a576b4e8c8426 138598
proteintargeting00maxi_0052.jpg
JPEG54.2 8bc791f69adbf5cfccfa3cea39286c84 57352
proteintargeting00maxi_0052.QC.jpg
09f228c221d9d64b02905bc4194b329e 140198
proteintargeting00maxi_0053.jpg
JPEG55.2 9a3c8e94bc5b19f9a602b6ecadc83243 57523
proteintargeting00maxi_0053.QC.jpg
83154dfd6a069b8921a8dfba68bcbf51 143297
proteintargeting00maxi_0054.jpg
JPEG56.2 c816b9d2d64f75d87e0c3593c73696cd 58802
proteintargeting00maxi_0054.QC.jpg
4822ee0edb6c9af75ac06602b98dbf5e 136310
proteintargeting00maxi_0055.jpg
JPEG57.2 90ac73cb9c7e988abd9ce3f59d35319a 56732
proteintargeting00maxi_0055.QC.jpg
efea091c66ba03e01ba0e5a7ce559886 134234
proteintargeting00maxi_0056.jpg
JPEG58.2 cfc401968bc9a170b01613a934442e3a 56802
proteintargeting00maxi_0056.QC.jpg
0c4a0689a1062d88264a1255ec0ba974 43080
proteintargeting00maxi_0057.jpg
JPEG59.2 1639c8a4f680221b34c489d16ce559d8 20594
proteintargeting00maxi_0057.QC.jpg
109747312e65e915f1202152dcc96255 116792
proteintargeting00maxi_0058.jpg
JPEG60.2 7b7902e555b83ddea9d77a365ff94e78 48902
proteintargeting00maxi_0058.QC.jpg
4d7b008fa018956dbd34ae0ea46119c2 135819
proteintargeting00maxi_0059.jpg
JPEG61.2 c6103d6e09c31b80d6759da8f187b602 53928
proteintargeting00maxi_0059.QC.jpg
e8a954ad280d0337c8624d8595459d08 120117
proteintargeting00maxi_0060.jpg
JPEG62.2 741fee1c2669f940b7a02464153af39c 51469
proteintargeting00maxi_0060.QC.jpg
7334d4970b55d5721ec5c5b70518c0fe 149984
proteintargeting00maxi_0061.jpg
JPEG63.2 939b739831cc71fb875c650b288471ad 60511
proteintargeting00maxi_0061.QC.jpg
6e1432429de63d1bb496103bf0f3a5f7 138625
proteintargeting00maxi_0062.jpg
JPEG64.2 801662b5ce22c123f6bcacf5b6765c50 60876
proteintargeting00maxi_0062.QC.jpg
e31dd5ddf048f7b40eabe6985ded1888 121210
proteintargeting00maxi_0063.jpg
JPEG65.2 7680f46412144c8222e4b4384dc57167 51702
proteintargeting00maxi_0063.QC.jpg
40f3c10bdb3086653d4892fc902de684 140283
proteintargeting00maxi_0064.jpg
JPEG66.2 a528a0b39507aac329b78d16e099fcc3 59106
proteintargeting00maxi_0064.QC.jpg
0c39041955d0db046870555636060bf2 122903
proteintargeting00maxi_0065.jpg
JPEG67.2 573b15d2e6dc41ae452c0dc06a5adcbe 42679
proteintargeting00maxi_0065.QC.jpg
8fb2dad5b8c19751a69f36d33aa49ddb 140653
proteintargeting00maxi_0066.jpg
JPEG68.2 29f7702c2bc1246bd3e636ab47d0082d 59079
proteintargeting00maxi_0066.QC.jpg
dbe0e8bb72f0a1796d5195073f8ca2b0 155852
proteintargeting00maxi_0067.jpg
JPEG69.2 1010e682cb1c3aadc23ea1ab04259381 52691
proteintargeting00maxi_0067.QC.jpg
b4d85d2bb2391153d35a9b708861ab43 138890
proteintargeting00maxi_0068.jpg
JPEG70.2 2a248e429d0afb9913af6546a8b23086 57162
proteintargeting00maxi_0068.QC.jpg
94d5cc480d14d547a7b185d64571355b 127297
proteintargeting00maxi_0069.jpg
JPEG71.2 de9d3a6c28afa69d50faf138e06131de 55335
proteintargeting00maxi_0069.QC.jpg
edec5d4278d40307a42263b8807b03de 103524
proteintargeting00maxi_0070.jpg
JPEG72.2 e389cd5e685c89e97f1af91c4153f2f9 40159
proteintargeting00maxi_0070.QC.jpg
29acfd07c0f3df049f02efd469dcc486 136022
proteintargeting00maxi_0071.jpg
JPEG73.2 2e8423aeb8b1571f98db47bc064f6439 47745
proteintargeting00maxi_0071.QC.jpg
1f1f4b2f4be6918ce4f38155030d9e42 136433
proteintargeting00maxi_0072.jpg
JPEG74.2 55189e88ce978e933d6b1508047087c2 53383
proteintargeting00maxi_0072.QC.jpg
a1b32af67b4a6543de43123153b29ccd 124134
proteintargeting00maxi_0073.jpg
JPEG75.2 5c53ae434619eb3753ae68faa37b7636 44993
proteintargeting00maxi_0073.QC.jpg
f39f4e034b5e0403f1ded1b6a69e0f7a 132664
proteintargeting00maxi_0074.jpg
JPEG76.2 a0673edae8add197c6b193064685a0f3 54644
proteintargeting00maxi_0074.QC.jpg
3d4cfc08336b1498f7cfd8a085bf2edc 131685
proteintargeting00maxi_0075.jpg
JPEG77.2 5eef29924c542bb176df2e1ed3300c22 45229
proteintargeting00maxi_0075.QC.jpg
5481480ad0b3dc9d4b5897324d72096c 112580
proteintargeting00maxi_0076.jpg
JPEG78.2 d4e142e3fa0ab577c6a2dedb7208918b 43406
proteintargeting00maxi_0076.QC.jpg
2992f71844c60b6c027fb0b643a0ca64 143934
proteintargeting00maxi_0077.jpg
JPEG79.2 00213e3074d4be6766329aa51eee0d47 56389
proteintargeting00maxi_0077.QC.jpg
dbdadcf085a4a8dce374efb1e3c5d6bf 127731
proteintargeting00maxi_0078.jpg
JPEG80.2 99b94eb3aa3791ae65207e4ddb2f6f71 53983
proteintargeting00maxi_0078.QC.jpg
403c28602947c1043e629f99cb132d86 129125
proteintargeting00maxi_0079.jpg
JPEG81.2 02609e83c1c70df727d63dfe7335c322 45141
proteintargeting00maxi_0079.QC.jpg
042325166d10c7fd58f799bd3e30a853 78668
proteintargeting00maxi_0080.jpg
JPEG82.2 2f60fb3b51b49fa1c6e26fcdcd944211 32810
proteintargeting00maxi_0080.QC.jpg
2b8a27b05c700de5c840558f17e60cef 133507
proteintargeting00maxi_0081.jpg
JPEG83.2 37e38984d7e4b274f0672bf000c3c83a 54887
proteintargeting00maxi_0081.QC.jpg
d9f6480238deb00aad80afa16c00fec8 108258
proteintargeting00maxi_0082.jpg
JPEG84.2 4d35a9a736db99be856c12608205f70d 39967
proteintargeting00maxi_0082.QC.jpg
6c68439b783d0dd83b3984f8172617c3 134520
proteintargeting00maxi_0083.jpg
JPEG85.2 3e5f51378cc42fabc685343c2375d204 54112
proteintargeting00maxi_0083.QC.jpg
9af13ed28b0119c8be2e00ff07b071ba 151617
proteintargeting00maxi_0084.jpg
JPEG86.2 ca79c71cdee3cbc2fc1249d140438638 59206
proteintargeting00maxi_0084.QC.jpg
87aaf99e2da2aaa70f35a08c4137edc8 146937
proteintargeting00maxi_0085.jpg
JPEG87.2 1a930ce47f717e989b2718a889757368 60362
proteintargeting00maxi_0085.QC.jpg
1338dcbe6b05681342fa589aedd29eb1 106905
proteintargeting00maxi_0086.jpg
JPEG88.2 df31af50b1d7c3b3d075ba731e889a38 45445
proteintargeting00maxi_0086.QC.jpg
96908d87c76821c3bf4b6098e5704745 119685
proteintargeting00maxi_0087.jpg
JPEG89.2 947a9de81cc9ef946d3117fe9a3babec 48950
proteintargeting00maxi_0087.QC.jpg
4a2b2d177ee0aa0b674004d1156f646e 128754
proteintargeting00maxi_0088.jpg
JPEG90.2 3be4363e140a8ddae12a263a7a3f021d 50461
proteintargeting00maxi_0088.QC.jpg
d0523e797137b69bc06efbfcf0e9a720 147170
proteintargeting00maxi_0089.jpg
JPEG91.2 6b63a212b70e233765b1d5fb58c1f4a9 55007
proteintargeting00maxi_0089.QC.jpg
b99e5bf7d209ac222159674e2cfc2748 167423
proteintargeting00maxi_0090.jpg
JPEG92.2 47509a3beaadf62f993bd3fb4ae85507 61161
proteintargeting00maxi_0090.QC.jpg
90eba0b8de95f4584734a6ce803ed341 166323
proteintargeting00maxi_0091.jpg
JPEG93.2 32a266c540ba07d6e905012500ae497a 60931
proteintargeting00maxi_0091.QC.jpg
6f2615f49b9bf79172fb3a3a6bde09b1 171439
proteintargeting00maxi_0092.jpg
JPEG94.2 f9f5393d379f2740fa5a2a32e7b01bd5 62217
proteintargeting00maxi_0092.QC.jpg
d2c60b57962949d5320b1fa1c79643a5 166593
proteintargeting00maxi_0093.jpg
JPEG95.2 934446fef381b0ecc8492d65350809d7 60351
proteintargeting00maxi_0093.QC.jpg
346b3b45e77818385ab08bd287df8680 160891
proteintargeting00maxi_0094.jpg
JPEG96.2 785897b5782f520deeadc543a44c4461 59247
proteintargeting00maxi_0094.QC.jpg
bbb08d497da0b9e573164a9134e9ba3d 163897
proteintargeting00maxi_0095.jpg
JPEG97.2 f6a24b7f4ccf82c39fd23a0f718cc024 59594
proteintargeting00maxi_0095.QC.jpg
c9ea4e961995165b15e603a542a7858d 170823
proteintargeting00maxi_0096.jpg
JPEG98.2 efff1d0d9e5e2192e3b11af3b34c39d5 62237
proteintargeting00maxi_0096.QC.jpg
54e73188a064a300c35800b348f1ceb7 169384
proteintargeting00maxi_0097.jpg
JPEG99.2 c628d381b669c611b38709c7cfd563e6 62395
proteintargeting00maxi_0097.QC.jpg
6f383ed6c98541e0cccf6a00ed7cd38f 39268
proteintargeting00maxi_0098.jpg
JPEG100.2 64d275304c013d3d7b9f5c4538231f18 20168
proteintargeting00maxi_0098.QC.jpg
cfe82c2977bd778b21d0837f179e4688 101577
proteintargeting00maxi_0099.jpg
JPEG101.2 2bc8fcd5f469d94e0c70ed21b1e57508 40246
proteintargeting00maxi_0099.QC.jpg
70840141acb8d69b253dcaa62f7eb10b 113869
proteintargeting00maxi_0100.jpg
JPEG102.2 2a7db50a7abc83064c73cbfbb83e6af1 44192
proteintargeting00maxi_0100.QC.jpg
3baf26448d2482b5f579d8f76e857b7b 40281
proteintargeting00maxi_0101.jpg
JPEG103.2 3c06951eb48194e0e48c3753aa1ee0c7 21382
proteintargeting00maxi_0101.QC.jpg
THUMB1 imagejpeg-thumbnails eb3827989fe07e8d738a3c481c07094c 15853
proteintargeting00maxi_0000thm.jpg
THUMB2 56d4f467005a1ab7efdbc3da19bf084e 19952
proteintargeting00maxi_0001thm.jpg
THUMB3 51588fb509edfd2be55c8982521f5f54 19620
proteintargeting00maxi_0002thm.jpg
THUMB4 7e80082596bccb71c75e4be693f60d73 16374
proteintargeting00maxi_0003thm.jpg
THUMB5 cab83d2e16bfd6810beb3b0009f6e09c 23094
proteintargeting00maxi_0004thm.jpg
THUMB6 99c4eaca4a5ba91d21c38bd5ba2ae242 22413
proteintargeting00maxi_0005thm.jpg
THUMB7 87ec0f654a79e1ca9275fb89dc6a8897 22933
proteintargeting00maxi_0006thm.jpg
THUMB8 f9559d59178b0cd615c0c35ff7b59359 24313
proteintargeting00maxi_0007thm.jpg
THUMB9 28938cfc82c49c6834555419a6336480 23889
proteintargeting00maxi_0008thm.jpg
THUMB10 43e4aa027920a1e9a1ae8c9671d64f6c 26327
proteintargeting00maxi_0009thm.jpg
THUMB11 0b1c8de876ad09ffa4043762954871ed 25019
proteintargeting00maxi_0010thm.jpg
THUMB12 ef3df0fe99dde7f2422585c3d653c648 25345
proteintargeting00maxi_0011thm.jpg
THUMB13 a644d8860c5873723ee00de40de80730 25687
proteintargeting00maxi_0012thm.jpg
THUMB14 74ec5712f2c1ca7a515ea95e5259b917 20041
proteintargeting00maxi_0013thm.jpg
THUMB15 b7fd751a8c2a137c9a3499215fb77e5c 26363
proteintargeting00maxi_0014thm.jpg
THUMB16 d6fdada376d8503740f4ab38fc33967e 20485
proteintargeting00maxi_0015thm.jpg
THUMB17 6e8c42bd5f988d293ffb4b54dfca1b15 24837
proteintargeting00maxi_0016thm.jpg
THUMB18 46971c80b54b7174cbb12a0da854295a 25323
proteintargeting00maxi_0017thm.jpg
THUMB19 cb10ab04ef62737fc2fe8a52f156c232 24764
proteintargeting00maxi_0018thm.jpg
THUMB20 4172dc0823f0678794ef2c7a2dc90767 25136
proteintargeting00maxi_0019thm.jpg
THUMB21 bd69044cf4c024777f163aa097e19b66 24567
proteintargeting00maxi_0020thm.jpg
THUMB22 f0a0b6c046871802e964a0da731fffeb 26336
proteintargeting00maxi_0021thm.jpg
THUMB23 a8623f749175f53649189d78e7fefa31 26212
proteintargeting00maxi_0022thm.jpg
THUMB24 939779b8d17f5a15e2ab01a9409f4d3b 24696
proteintargeting00maxi_0023thm.jpg
THUMB25 01951de7d139f72e85f1a24c7f9adfe4 24799
proteintargeting00maxi_0024thm.jpg
THUMB26 ff194286ca54d931b53feb3b84a1055a 24972
proteintargeting00maxi_0025thm.jpg
THUMB27 17cdd5ed34e2fec0b21fdeb438eb0522 16186
proteintargeting00maxi_0026thm.jpg
THUMB28 fb493e3bbb18e5d4b6fcbc91014fbcb7 34312
proteintargeting00maxi_0056athm.jpg
THUMB29 efa450d21affb539e80370dd33705163 25127
proteintargeting00maxi_0027thm.jpg
THUMB30 c2a2b9110e67a7a968157cc9c7cc2b8f 26133
proteintargeting00maxi_0028thm.jpg
THUMB31 0545384b6eca7edc819f4a2beb3e3d78 27043
proteintargeting00maxi_0029thm.jpg
THUMB32 a2fe6a957228bfcd5cfbd4883abb50fe 24118
proteintargeting00maxi_0030thm.jpg
THUMB33 cc5982812f3135cdd8443aeb656ee695 27215
proteintargeting00maxi_0031thm.jpg
THUMB34 ef4c13249c665888299cc1b2aea50968 26504
proteintargeting00maxi_0032thm.jpg
THUMB35 ec41dff20f93af4a4c65d44aef520316 26648
proteintargeting00maxi_0033thm.jpg
THUMB36 7a62a7bd7e659b2d1c6d8604a41f191f 24184
proteintargeting00maxi_0034thm.jpg
THUMB37 6454878b93f68a011b7a3bbee3f7ba7f 26277
proteintargeting00maxi_0035thm.jpg
THUMB38 c947a425e3b0ac79f02a85100fa1be65 20338
proteintargeting00maxi_0036thm.jpg
THUMB39 53a90b6deac72dd3fb33f376b0c10907 26169
proteintargeting00maxi_0037thm.jpg
THUMB40 b19cf4e17469cd0897a4bf1c1696b993 23394
proteintargeting00maxi_0038thm.jpg
THUMB41 fb4e323c65ec7a6a4cd83faf4c4c7935 16652
proteintargeting00maxi_0039thm.jpg
THUMB42 1e26c26ce62588ce452fd3fb2ee65847 26921
proteintargeting00maxi_0040thm.jpg
THUMB43 e0aabd0ec9abd25cbcd20439c7ed9e81 21175
proteintargeting00maxi_0041thm.jpg
THUMB44 7b60861a61fa2090da54a5cde561f4ba 26022
proteintargeting00maxi_0042thm.jpg
THUMB45 efa2f2d31df6cf62b6174074bd3401fc 21566
proteintargeting00maxi_0043thm.jpg
THUMB46 fd65084a3c2f7e62af2c6223acf89bfb 26745
proteintargeting00maxi_0044thm.jpg
THUMB47 b7647c4f8bd78c02d411501854144833 23406
proteintargeting00maxi_0045thm.jpg
THUMB48 6164aeb12a2e90f2632802921a0a61ee 25541
proteintargeting00maxi_0046thm.jpg
THUMB49 048318d59b751bd9b34f6fe92eedc715 22060
proteintargeting00maxi_0047thm.jpg
THUMB50 fcc3a4f22c6e491787ec415786c8bb3d 26380
proteintargeting00maxi_0048thm.jpg
THUMB51 47d384ab31352cf6fc0da0f6964f67ef 16916
proteintargeting00maxi_0049thm.jpg
THUMB52 965b690a57c113755ecd028454b0413b 22026
proteintargeting00maxi_0050thm.jpg
THUMB53 d948181929397f16eb896c187c061017 25768
proteintargeting00maxi_0051thm.jpg
THUMB54 4f47d8688fde915e9eb8313b63884046 27248
proteintargeting00maxi_0052thm.jpg
THUMB55 4944eb74fcc1c6cdf02e794b9cf797e4 26835
proteintargeting00maxi_0053thm.jpg
THUMB56 ea5e94f221ae34aa442113502b6705c5 27231
proteintargeting00maxi_0054thm.jpg
THUMB57 8b8ed8399d375c5217766b364d7a4f0a 26751
proteintargeting00maxi_0055thm.jpg
THUMB58 9893dee9fcafab27a87636585c5db88f 26802
proteintargeting00maxi_0056thm.jpg
THUMB59 0cf6ec252ac2351aa399942300a18379 14993
proteintargeting00maxi_0057thm.jpg
THUMB60 dbecdd8174767da852ea4a4b3b82adc9 24730
proteintargeting00maxi_0058thm.jpg
THUMB61 9441a3e71fddee323d9ef2eed5dae004 25964
proteintargeting00maxi_0059thm.jpg
THUMB62 7bc16000dfc94560c90c33941441e20e 24713
proteintargeting00maxi_0060thm.jpg
THUMB63 86284dbbcce3194e9bd97559d0879b96 26466
proteintargeting00maxi_0061thm.jpg
THUMB64 e8c1a7aa77ecfdc50de0df347589d3a4 26248
proteintargeting00maxi_0062thm.jpg
THUMB65 3664f8a5863553b9fac2b30761410188 24785
proteintargeting00maxi_0063thm.jpg
THUMB66 8b6bfd223f42ee9b05f05b6c807bc3b2 26773
proteintargeting00maxi_0064thm.jpg
THUMB67 880ae8f83ac2f4b879b3f298492c0177 20899
proteintargeting00maxi_0065thm.jpg
THUMB68 ffadd1fcc4aa13a01bb8f62e2f3e1b5e 26763
proteintargeting00maxi_0066thm.jpg
THUMB69 c655439387ff985bb7b5265f0fca62cb 24547
proteintargeting00maxi_0067thm.jpg
THUMB70 5372062333bb76927cf8411cd975d59d 26872
proteintargeting00maxi_0068thm.jpg
THUMB71 db02e7491e21faf4de2814a2c003bd66 25564
proteintargeting00maxi_0069thm.jpg
THUMB72 4729cef2479a49eb9a77e763784b4233
proteintargeting00maxi_0070thm.jpg
THUMB73 67bc0360c02067a83b3d1af99d73dbda 22748
proteintargeting00maxi_0071thm.jpg
THUMB74 144f64e0a98bf0b8ea68595dd3fc415d 25368
proteintargeting00maxi_0072thm.jpg
THUMB75 b7f8651384108683dfb7031a2702041b 22538
proteintargeting00maxi_0073thm.jpg
THUMB76 dd62e6f4a899ec2b654cd311caafe27a 25508
proteintargeting00maxi_0074thm.jpg
THUMB77 f57fbe322582cb7173ca85a6557630a3 22094
proteintargeting00maxi_0075thm.jpg
THUMB78 d90301657a04fb217852a16b5b34afda 22824
proteintargeting00maxi_0076thm.jpg
THUMB79 b6facc6e89a851565caef90c10588d1a 26490
proteintargeting00maxi_0077thm.jpg
THUMB80 60345c93c47ada52fd5be434fc6dd689 25263
proteintargeting00maxi_0078thm.jpg
THUMB81 0c27814694b84498c41f3817eb405c43 21746
proteintargeting00maxi_0079thm.jpg
THUMB82 f2b18dabbd0fb7667f5e2a960e7a6d6e 18846
proteintargeting00maxi_0080thm.jpg
THUMB83 10890f5f93ea7cb7b06bae530fb0261b 26318
proteintargeting00maxi_0081thm.jpg
THUMB84 053ba57776156872df11658b5946193b 20603
proteintargeting00maxi_0082thm.jpg
THUMB85 a8bcf93196323e5cfe7fdfed8a2b0f53 26158
proteintargeting00maxi_0083thm.jpg
THUMB86 08d319e59806fa51e0228a968ab5d62f 27358
proteintargeting00maxi_0084thm.jpg
THUMB87 fc1c16d024a3318560ffbe8117d34a75 27145
proteintargeting00maxi_0085thm.jpg
THUMB88 1ea7ed38a8e64051f3a49c9c07a191cb 22430
proteintargeting00maxi_0086thm.jpg
THUMB89 6abfc62ecd6c75a1f93b1c6da690a6e9 24562
proteintargeting00maxi_0087thm.jpg
THUMB90 245403f1c4da9e823245346a696a7bd2 25453
proteintargeting00maxi_0088thm.jpg
THUMB91 85840c2abb8d86c64c290690906f3686 25599
proteintargeting00maxi_0089thm.jpg
THUMB92 f383bd1c0a652bf18e60c0475109432c 27984
proteintargeting00maxi_0090thm.jpg
THUMB93 80f24e468dcf7b39f6865f35912dba6c 27610
proteintargeting00maxi_0091thm.jpg
THUMB94 be44b2477318c7eb8e93f043087c2277 28073
proteintargeting00maxi_0092thm.jpg
THUMB95 10ab975c59b8d3158feb68312d3e74af 28060
proteintargeting00maxi_0093thm.jpg
THUMB96 0e50740f2296c4de113c02a353762adb 27786
proteintargeting00maxi_0094thm.jpg
THUMB97 49741976ecf71693bd63e1e224962ede 28184
proteintargeting00maxi_0095thm.jpg
THUMB98 65c8b67f91e119e830109f641f38f60a 28183
proteintargeting00maxi_0096thm.jpg
THUMB99 3ef93837f7473d39560537fb60b6bb81 28281
proteintargeting00maxi_0097thm.jpg
THUMB100 6be4116bf64737cb85ff2b3d2ea5d608 14161
proteintargeting00maxi_0098thm.jpg
THUMB101 b4c5926a3ceaf68788d065d1e947c565 21538
proteintargeting00maxi_0099thm.jpg
THUMB102 004467f950c9e145f3b2157b12b86d6e 22301
proteintargeting00maxi_0100thm.jpg
THUMB103 27202a38e01b648a8eb43f06349b8d42 14614
proteintargeting00maxi_0101thm.jpg
TXT1 textplain 1e9baf0435690172c9bdd3d60c0bac3d 433
proteintargeting00maxi_0000.txt
TXT2 1efc05d9b4fb396585ac7d5c98e5abd1 1009
proteintargeting00maxi_0001.txt
TXT3 b0b0da6120b43b33ab7d60ff7c05c365 2913
proteintargeting00maxi_0002.txt
TXT4 a68fb07298f25bd4ac2da17e1bae0a70 629
proteintargeting00maxi_0003.txt
TXT5 5dbc3fd54af49de8805bfc2de626013b 1545
proteintargeting00maxi_0004.txt
TXT6 3a5cc5a0a2c19b61cd5085d6a41f9f1a 1281
proteintargeting00maxi_0005.txt
TXT7 94511d5c6fca232d91206ab27a5c53ed 1593
proteintargeting00maxi_0006.txt
TXT8 57640300459bb7caf88018c294c6cc5e 1745
proteintargeting00maxi_0007.txt
TXT9 27fa00878791fe1581872ad1a35139ca 1609
proteintargeting00maxi_0008.txt
TXT10 a0786ad42feb78df22d19c816479b049 2020
proteintargeting00maxi_0009.txt
TXT11 deb16ef2a4303de1e4472d5f34e107e5 1787
proteintargeting00maxi_0010.txt
TXT12 a5b1a37da3b9cea3fb5ce54c18ad606b 1876
proteintargeting00maxi_0011.txt
TXT13 2a0d5144ce7ac8d3b83dce0048dc3b1d 1904
proteintargeting00maxi_0012.txt
TXT14 c0128fd0d9a40ea4a7be351fb6a8d90e 623
proteintargeting00maxi_0013.txt
TXT15 9f90fabdb3ba34a10c9e9a33ee81dc10 1833
proteintargeting00maxi_0014.txt
TXT16 c806e052afc2e3104431cba272fa8f10 1466
proteintargeting00maxi_0015.txt
TXT17 701f5930c2b015bb951b47880aee4b8d 1797
proteintargeting00maxi_0016.txt
TXT18 efbeb84387464c6332d00005aeaa9fb8 1819
proteintargeting00maxi_0017.txt
TXT19 a4aa2f990f3694eb47105c55436868e9 1758
proteintargeting00maxi_0018.txt
TXT20 7cd6ef6773ba16bb1e9b07485dc12a70 1782
proteintargeting00maxi_0019.txt
TXT21 a508ac28c71f3b18c7ce2e7420d00b37 1696
proteintargeting00maxi_0020.txt
TXT22 c91317cc5acd828d80495cf2d43d549b 1935
proteintargeting00maxi_0021.txt
TXT23 b64ddef112b9862b35e30a0c9cbc8be1 1993
proteintargeting00maxi_0022.txt
TXT24 6b08426a11f4241815b71a42eaa09dde 1683
proteintargeting00maxi_0023.txt
TXT25 a8f1984b4fc7da6b2fda0ea200d6f870 1738
proteintargeting00maxi_0024.txt
TXT26 83eeed89085a6b0ab3f3a99dbde3dc81 1823
proteintargeting00maxi_0025.txt
TXT27 2e88a0970f614ee192b156a6e5748101 591
proteintargeting00maxi_0026.txt
TXT28 da3c3b0a8381b18823a9d88014f2ccb9 1737
proteintargeting00maxi_0056a.txt
TXT29 0eba5077430a7833ea43afbe36871e0c 1648
proteintargeting00maxi_0027.txt
TXT30 58b0a80bea20c34cb6d1c1f1620489c8 1853
proteintargeting00maxi_0028.txt
TXT31 497a99fe59cf1a3b1ef3100a830f0d04 1911
proteintargeting00maxi_0029.txt
TXT32 700774e23be7680124ea914d61859288 1551
proteintargeting00maxi_0030.txt
TXT33 509205eb376f5cea8996e0d777ca5584 1812
proteintargeting00maxi_0031.txt
TXT34 06352e752ce2723293e5fb5db28884b7 1801
proteintargeting00maxi_0032.txt
TXT35 727bd871e00db1c11b539d0c7e940c15 1717
proteintargeting00maxi_0033.txt
TXT36 1fb992f1e1b194d4c55cde4b1ca6a71b 1533
proteintargeting00maxi_0034.txt
TXT37 bcf9917daa898aa45a955660ac3aa455 1805
proteintargeting00maxi_0035.txt
TXT38 0998c7c956501a2821cd98f9441a2ba6 1584
proteintargeting00maxi_0036.txt
TXT39 0fe7ba46dd106e0725276bd5f96e7bc5 1792
proteintargeting00maxi_0037.txt
TXT40 4a5ee803c85be616d933c7952b6376c2 2010
proteintargeting00maxi_0038.txt
TXT41 c6f09a1dde8108def8c65d459fe6b3b5 553
proteintargeting00maxi_0039.txt
TXT42 f066ae8d56391fa831425b361cd276b8 1840
proteintargeting00maxi_0040.txt
TXT43 10a40e035e0846502c7ac8d1a26b35be 1673
proteintargeting00maxi_0041.txt
TXT44 973c1f43777f4d23b383923fa9bd649b 1803
proteintargeting00maxi_0042.txt
TXT45 9f6889f530b818eb4683f7424b86f719 1448
proteintargeting00maxi_0043.txt
TXT46 36fed44c15d3597ba6550ff9f04c84ef 1899
proteintargeting00maxi_0044.txt
TXT47 331905259ea83cc9061fcbe9bc027496 1997
proteintargeting00maxi_0045.txt
TXT48 edc4bc4298e5a57742086063053476ff
proteintargeting00maxi_0046.txt
TXT49 7dacfe1c1b4c110d246f7eb896bd5664 2004
proteintargeting00maxi_0047.txt
TXT50 1dc6c877a7cf879456c275e54be2dea8 1827
proteintargeting00maxi_0048.txt
TXT51 a6e84466da7e985d9fd5314f2a7d54fe 776
proteintargeting00maxi_0049.txt
TXT52 8c024c23f257b678793dfd32c0260988 1665
proteintargeting00maxi_0050.txt
TXT53 f487b6f8a5139cccd03a88d907f0a197 1798
proteintargeting00maxi_0051.txt
TXT54 8a082655bc2a68b0c02487ee3660ca79 1896
proteintargeting00maxi_0052.txt
TXT55 b76b9cfe89e3fd0f5d4bd9f930b7f552
proteintargeting00maxi_0053.txt
TXT56 0501a33b3f06ee042cf359ae0a057f70 1966
proteintargeting00maxi_0054.txt
TXT57 bbe2a97830685985f13dc0d0f89493b7 1922
proteintargeting00maxi_0055.txt
TXT58 c65718a50284f960e27c87f5933d3a63
proteintargeting00maxi_0056.txt
TXT59 cf000ad4631fb78b8f1a1eb3fc22a1ba 354
proteintargeting00maxi_0057.txt
TXT60 6d56567f2be7657752ac18a13ee6cc2d 1606
proteintargeting00maxi_0058.txt
TXT61 0c127ed1df515870ee47fe5c6bd7bf59 1829
proteintargeting00maxi_0059.txt
TXT62 dc438354bdc46cde51f35f7fae21958e 1532
proteintargeting00maxi_0060.txt
TXT63 8e07676e3d389a7720ada8d11e1714f3 1725
proteintargeting00maxi_0061.txt
TXT64 659f35ccfaa4be01c96311b0f49226ef 1771
proteintargeting00maxi_0062.txt
TXT65 ca141f84949d7a20108f48f8bb35fa09 1599
proteintargeting00maxi_0063.txt
TXT66 79497e6f138f2a58d8149600d2204e3d 1894
proteintargeting00maxi_0064.txt
TXT67 50508c41bf697bdec04b6c17c5e391b2 1924
proteintargeting00maxi_0065.txt
TXT68 cad6e9c2783e9cfa5efbb0036f0cfaa1 1856
proteintargeting00maxi_0066.txt
TXT69 7c9aff906c5092f7f2c85fc1e1b68a14 2292
proteintargeting00maxi_0067.txt
TXT70 886d38f2dafaab3c17edbb1c5f10964e 1909
proteintargeting00maxi_0068.txt
TXT71 ef7a6e3e1db9666bcb5d40982908ffa3 1755
proteintargeting00maxi_0069.txt
TXT72 af180a6d318b15496139804f99ee3db8 1272
proteintargeting00maxi_0070.txt
TXT73 63ceee9a7ac825439753d914af8753f4 1892
proteintargeting00maxi_0071.txt
TXT74 d031d67cd60be77bf092534afbc6595d 1754
proteintargeting00maxi_0072.txt
TXT75 b4c50462b7c7a628c4131549b419879f 1730
proteintargeting00maxi_0073.txt
TXT76 bd2dbed70f5542bb6b86699bf1e3b06c 1814
proteintargeting00maxi_0074.txt
TXT77 102421c5c0ed0dcd0b117c98e9d80455 1701
proteintargeting00maxi_0075.txt
TXT78 78b16fcd088edeb85e32c53a705d5d2a 1602
proteintargeting00maxi_0076.txt
TXT79 f9b2ccf5a99de21bb8ebd2f03fb851aa
proteintargeting00maxi_0077.txt
TXT80 d7002be9f3c2e52ac23a1b433cca6c74 1724
proteintargeting00maxi_0078.txt
TXT81 5ce0f6430a473d03e1aaa3024d55c8c8 1670
proteintargeting00maxi_0079.txt
TXT82 650469819f860fed6a404c607d3b66d4 1079
proteintargeting00maxi_0080.txt
TXT83 9886a7d1a6e2f267337c9d6fe558f790 1750
proteintargeting00maxi_0081.txt
TXT84 616568851e437a3e6e34ac8dc8c7ae7b 1042
proteintargeting00maxi_0082.txt
TXT85 99ed7de256f9989e7cf9e5e942224ec9 1842
proteintargeting00maxi_0083.txt
TXT86 a3395ee0163834c8a5ce1f397eb7fd53 1955
proteintargeting00maxi_0084.txt
TXT87 26271cb7b6f0cbe71593d60c3cc00526 1975
proteintargeting00maxi_0085.txt
TXT88 9ad6c922aa5dbef83c715b54fbd30cb0 1174
proteintargeting00maxi_0086.txt
TXT89 0bd1969a1dff961f6ef5be2dbd749272 1651
proteintargeting00maxi_0087.txt
TXT90 28af79df2cff2ce321186c51672b74fd 1681
proteintargeting00maxi_0088.txt
TXT91 9bbde7e751a26b0642288d00d871f705 2128
proteintargeting00maxi_0089.txt
TXT92 fd35debeec28231d6d207e9b88dab3e5
proteintargeting00maxi_0090.txt
TXT93 3d2731d9a577d327b6c2ae437f748023 2333
proteintargeting00maxi_0091.txt
TXT94 e07f3bfc41945f5d4426e5a3358bfe10
proteintargeting00maxi_0092.txt
TXT95 d1d1e1b1ebeb5774d590a63a410c1c08 2349
proteintargeting00maxi_0093.txt
TXT96 42741926b2a3b47e9ba09d10256bd04e 2342
proteintargeting00maxi_0094.txt
TXT97 1be0479a1e9fdb3df38149497a5653fb 2366
proteintargeting00maxi_0095.txt
TXT98 7746dbeaec4a00446739cd9595fb95f8 2405
proteintargeting00maxi_0096.txt
TXT99 f9a15466dd6605cb59999ec8bec0ed4e 2428
proteintargeting00maxi_0097.txt
TXT100 fa45c13352705677d62d31be471eeb24 293
proteintargeting00maxi_0098.txt
TXT101 b93cb0da030bb26e3dc2abd9e537b680 1088
proteintargeting00maxi_0099.txt
TXT102 c11f35fde3774068b170a0fc81e09cfd 1945
proteintargeting00maxi_0100.txt
TXT103 b71ee40d77b126b70f768b7360fd6852 416
proteintargeting00maxi_0101.txt
PRO1 textx-pro dc67faacc43fe9c16fc0ae0c13a5f73d 8373
proteintargeting00maxi_0000.pro
PRO2 b3e9757774bedca1b4fd42c508010579 25892
proteintargeting00maxi_0001.pro
PRO3 7fdecb11c14a7e810c6325b29c36d57d 64011
proteintargeting00maxi_0002.pro
PRO4 ad202b639da5598289a1dafb22ef0dd8 15982
proteintargeting00maxi_0003.pro
PRO5 0c99ab67a599cfb92a6bb86765fd6070 37000
proteintargeting00maxi_0004.pro
PRO6 5c7a42dfca4a7351fe19bf74fcb4dbbf 33422
proteintargeting00maxi_0005.pro
PRO7 6b82bd95da598d843b81de7358048a59 39519
proteintargeting00maxi_0006.pro
PRO8 31d4305966400251ea6e4413b61f3fbf 44796
proteintargeting00maxi_0007.pro
PRO9 e961076c9b41af4459876f717b9515e5 42275
proteintargeting00maxi_0008.pro
PRO10 93445846fd922c9802875907c85c0bf6 53193
proteintargeting00maxi_0009.pro
PRO11 1a13415985fdb8685b630504fb987671 46388
proteintargeting00maxi_0010.pro
PRO12 41bca2e11c0795f4cd09aa973b388a11 49411
proteintargeting00maxi_0011.pro
PRO13 ad50d57eb62a0b378127a2c594b6ef41 49859
proteintargeting00maxi_0012.pro
PRO14 cd4bfbf65b4df438c7bde53c4ddfb492 8672
proteintargeting00maxi_0013.pro
PRO15 ca9e35d85b11c3ed69f2245832d73d82 48052
proteintargeting00maxi_0014.pro
PRO16 1c70e9c23c64a48a6084921e7d05e0df 34180
proteintargeting00maxi_0015.pro
PRO17 a68af85f09d3e67ac2c0c79f06e6fd34 47340
proteintargeting00maxi_0016.pro
PRO18 48a57226ca6b335f342849e75aa1bdd0 47732
proteintargeting00maxi_0017.pro
PRO19 75350a54b5874454ae5d38636a42b716 45952
proteintargeting00maxi_0018.pro
PRO20 10423c2f2e0b39d33b7c7ba0e8cc80c2 46992
proteintargeting00maxi_0019.pro
PRO21 8685adf8eb653afcb39893cf7f31f06e 44343
proteintargeting00maxi_0020.pro
PRO22 5b38bf8f943a26260661f5536e6810a0 51127
proteintargeting00maxi_0021.pro
PRO23 9bb2c45ff51ecc9d0734bb5e3d20a64e 52625
proteintargeting00maxi_0022.pro
PRO24 f743396e6959d7360df3c94b92a79170 43040
proteintargeting00maxi_0023.pro
PRO25 f0b3b08fb9ec9622242a00ff41511345 45514
proteintargeting00maxi_0024.pro
PRO26 257767211ccad6d12b492f3f736cb061 47802
proteintargeting00maxi_0025.pro
PRO27 a3eb60dbf809c2c2f65b859b648cfe7b 13061
proteintargeting00maxi_0026.pro
PRO28 552b33eb58d854539b1f1f8fdfd96d94 45362
proteintargeting00maxi_0056a.pro
PRO29 149414b1598961ce5bbf69327fdbd7f4 42411
proteintargeting00maxi_0027.pro
PRO30 4b7fe3345cec47cb9b77542d604b1abd 47678
proteintargeting00maxi_0028.pro
PRO31 9f82e1a1157789c408fff37208c1944f 50233
proteintargeting00maxi_0029.pro
PRO32 b1799dda309cf18925beeb73094d20bc 39474
proteintargeting00maxi_0030.pro
PRO33 01bf58ca4816217e4ae0cc52dab1e194 47712
proteintargeting00maxi_0031.pro
PRO34 e5de941eee0c798529c878b0fc5541ff 47411
proteintargeting00maxi_0032.pro
PRO35 f00f8ff3c8468f430f6eda61675e4973 45447
proteintargeting00maxi_0033.pro
PRO36 952fe116879c01eb841eb8a14895bf5d 39157
proteintargeting00maxi_0034.pro
PRO37 db83fedd2e98c5f99b5fe945e97ba443 47582
proteintargeting00maxi_0035.pro
PRO38 976cb95ed450c32f77fdb65f6ae7646e 33918
proteintargeting00maxi_0036.pro
PRO39 4b8df03e53a054484899ee195c6b4068 47169
proteintargeting00maxi_0037.pro
PRO40 3018f3d95af2cd8bea9b50fb50c70f7b
proteintargeting00maxi_0038.pro
PRO41 d7256c42293a624524468d593d4a1bb8 13063
proteintargeting00maxi_0039.pro
PRO42 4e9424750b9aeb3a38af4fc47d37cafd 48537
proteintargeting00maxi_0040.pro
PRO43 d6552bd13203d0377315d37de6fd8b7d 36469
proteintargeting00maxi_0041.pro
PRO44 0d87d07859053a4d032fdddd0b5b6db2 47456
proteintargeting00maxi_0042.pro
PRO45 cc19866e2a994bdf6114a5fda0652b1c 34176
proteintargeting00maxi_0043.pro
PRO46 75d9ff4432a3b841b7e0ab3cb0abbcde 50190
proteintargeting00maxi_0044.pro
PRO47 a10c1fbcea4826e369ce023999d840f9 48860
proteintargeting00maxi_0045.pro
PRO48 cd1fffec0477b0665f9aa98b1c603448 45655
proteintargeting00maxi_0046.pro
PRO49 ba5158c6e691d717234ee6014e0ed48d 42835
proteintargeting00maxi_0047.pro
PRO50 b552f0b4cd2c41efc3835f0ab321c108 48166
proteintargeting00maxi_0048.pro
PRO51 2f6e785eb452e1e5f32cc5b2556d181a 17053
proteintargeting00maxi_0049.pro
PRO52 30ba6a86d8cff83481484d79bdf5b7de 40356
proteintargeting00maxi_0050.pro
PRO53 8312e73a3b875097bf598eeeb07728c1 46426
proteintargeting00maxi_0051.pro
PRO54 ddd0c0e814ab636d6f8d0f511de5c353
proteintargeting00maxi_0052.pro
PRO55 8d33dd8d9aa6daf5e77b659087c4aa98 50591
proteintargeting00maxi_0053.pro
PRO56 2649215b7411e17b486ed37435db53be 51957
proteintargeting00maxi_0054.pro
PRO57 96571f5a0eec766e355cec9daed9b7b5 50623
proteintargeting00maxi_0055.pro
PRO58 c4e6e57f7f80c211a27468073bc51e36 50799
proteintargeting00maxi_0056.pro
PRO59 f6800d0605ea64752c5f13f5b9e64412 6990
proteintargeting00maxi_0057.pro
PRO60 f61719b792d189656cda9705d9367a86 41120
proteintargeting00maxi_0058.pro
PRO61 e262838897809e3aa05e63f0ede2e503 47206
proteintargeting00maxi_0059.pro
PRO62 88cee152d031d20fc4bda54f3e4a3c41 39266
proteintargeting00maxi_0060.pro
PRO63 9c06aedcce509428f0243815d8a6c370 45066
proteintargeting00maxi_0061.pro
PRO64 571fb5da12128c429e5299feeed47591 46505
proteintargeting00maxi_0062.pro
PRO65 fd56ee85243b812a937e2ff2640c829f 40695
proteintargeting00maxi_0063.pro
PRO66 dc2eeb182293c4ac1411126ddc9eb0b2 49832
proteintargeting00maxi_0064.pro
PRO67 e1fc18e0bebd4bd1d391f78b72f07ce5 42022
proteintargeting00maxi_0065.pro
PRO68 355cff587216ac9573d96879d013adaa 48832
proteintargeting00maxi_0066.pro
PRO69 eb08447968ce6ffaa71e84c801c3364e 51409
proteintargeting00maxi_0067.pro
PRO70 06a04d7310b8f7b4760a484cb7b6236e 50491
proteintargeting00maxi_0068.pro
PRO71 353da731ba6b91a6ec3a9ebe4218eb0c 45862
proteintargeting00maxi_0069.pro
PRO72 eeaf9ea0fe1762d3eeeb04c19329be81 30357
proteintargeting00maxi_0070.pro
PRO73 8a66aadab0ef448148a066a2920363a7 42737
proteintargeting00maxi_0071.pro
PRO74 c913c84bb154efae627a3c42a50147e9 46055
proteintargeting00maxi_0072.pro
PRO75 19c351dbd8d6bd8e8809fb49c667a2ba 33789
proteintargeting00maxi_0073.pro
PRO76 e19bf6c0759cef833824f2e6b7f2a147 47704
proteintargeting00maxi_0074.pro
PRO77 93a92675018fc4a9f7876a6eb1fe0a47 41682
proteintargeting00maxi_0075.pro
PRO78 dced8a22a5a8feb13a160c7e0bc14af3 28639
proteintargeting00maxi_0076.pro
PRO79 ea46d94a983f08adb6ec7fb416ea9a7a 50225
proteintargeting00maxi_0077.pro
PRO80 87f698da5f44555503733900620d2972 45120
proteintargeting00maxi_0078.pro
PRO81 df103dd9b135c6a35c6de1407d8d3c1a 41043
proteintargeting00maxi_0079.pro
PRO82 afcb4f312b5c5aef92ded055f815e155
proteintargeting00maxi_0080.pro
PRO83 867e65a0c96c42e87e73fc8e0b78908a 46006
proteintargeting00maxi_0081.pro
PRO84 5a6aedb9d802e8de97968cbbcc93eb70 25016
proteintargeting00maxi_0082.pro
PRO85 9e90ee5244829cb02a2a270b3b54eece 47692
proteintargeting00maxi_0083.pro
PRO86 8f861e124db8ccd7e900bdd62351f910 51709
proteintargeting00maxi_0084.pro
PRO87 87e70053392bb946cd74a90516f4f88f 52291
proteintargeting00maxi_0085.pro
PRO88 b0f5a8ffee610729d4fe24f31d582615 30565
proteintargeting00maxi_0086.pro
PRO89 1b32aa9a9b83fc5d3ab040ec1e478f9e 41696
proteintargeting00maxi_0087.pro
PRO90 283c1fec9a645b424a1fa78103e20796 43987
proteintargeting00maxi_0088.pro
PRO91 dc9c5b1d0d6a79663170479237027a74 55490
proteintargeting00maxi_0089.pro
PRO92 cd5f578746a17ba417848bb1b0f96b63 63159
proteintargeting00maxi_0090.pro
PRO93 fcf76f8bda47d8b25a357010ffaaeb75 60914
proteintargeting00maxi_0091.pro
PRO94 533b0f5bd0463585e55910faecf7094b 62994
proteintargeting00maxi_0092.pro
PRO95 52b9bf01f836545f454b78abc339b204 61511
proteintargeting00maxi_0093.pro
PRO96 6ef9c064bd8f3d24badf0cf2bb06adb4 61363
proteintargeting00maxi_0094.pro
PRO97 a81d1252819abdd74ad3b6b4d1e037ba 61925
proteintargeting00maxi_0095.pro
PRO98 2627e06578de98e86626bac834061ef8 62962
proteintargeting00maxi_0096.pro
PRO99 067267e21e1c660bca92e0f80e6762a2 63677
proteintargeting00maxi_0097.pro
PRO100 0cce3c92a805da09c72c866c9c2f8266 5580
proteintargeting00maxi_0098.pro
PRO101 adbf0a1cc3b4f851e20232f6560701ed 27999
proteintargeting00maxi_0099.pro
PRO102 8167d32d78575c7dbd9391bab4a7a902 38999
proteintargeting00maxi_0100.pro
PRO103 7f828af3808b0c2bf69637133e7aee9e 7400
proteintargeting00maxi_0101.pro
PDF1 applicationpdf 97bc7b4ca7ebafd14cfbb3cc55dff970 4697290
proteintargeting00maxi.pdf
METS2 unknownx-mets 765bfa454ecc12a0b8accc1d31a3363f 114966
AA00030021_00001.mets
METS:structMap STRUCT1 physical
METS:div DMDID ADMID ORDER 0 main
PDIV1 1 Title Page
PAGE1 i
METS:fptr FILEID
PDIV2 Acknowledgments 2 Section
PAGE2 ii
PDIV3 3 Table Contents
PAGE3 iii
PDIV4 Key to Abbreviations 4
PAGE4 iv
PDIV5 5 Abstract
PAGE5 v
PAGE6 vi
PDIV6 Chapter 1. Literature review 6
PAGE7
PAGE8
PAGE9
PAGE10
PAGE11
PAGE12
PAGE13 7
PAGE14 8
PAGE15 9
PAGE16 10
PAGE17 11
PAGE18 12
PAGE19 13
PAGE20 14
PAGE21 15
PAGE22 16
PAGE23 17
PAGE24 18
PAGE25 19
PAGE26 20
PAGE27 21
PDIV7 2. the pathway
PAGE28 52
PAGE29 22
PAGE30 23
PAGE31 24
PAGE32 25
PAGE33 26
PAGE34 27
PAGE35 28
PAGE36 29
PAGE37 30
PAGE38 31
PAGE39 32
PAGE40 33
PAGE41 34
PAGE42 35
PAGE43 36
PAGE44 37
PAGE45 38
PAGE46 39
PAGE47 40
PAGE48 41
PAGE49 42
PAGE50 43
PAGE51 44
PAGE52 45
PAGE53 46
PAGE54 47
PAGE55 48
PAGE56 49
PAGE57 50
PAGE58 51
PAGE59 53
PDIV8 3.
PAGE60 54
PAGE61 55
PAGE62 56
PAGE63 57
PAGE64 58
PAGE65 59
PAGE66 60
PAGE67 61
PAGE68 62
PAGE69 63
PAGE70 64
PAGE71 65
PAGE72 66
PAGE73 67
PAGE74 68
PAGE75 69
PAGE76 70
PAGE77 71
PAGE78 72
PAGE79 73
PAGE80 74
PAGE81 75
PAGE82 76
PAGE83 77
PAGE84 78
PAGE85 79
PAGE86 80
PAGE87 81
PAGE88 82
PDIV9 4. Summary conclusions
PAGE89 83
PAGE90 84
PDIV10 References
PAGE91 85
PAGE92 86
PAGE93 87
PAGE94 88
PAGE95 89
PAGE96 90
PAGE97 91
PAGE98 92
PAGE99 93
PAGE100 94
PDIV11 Biographical sketch
PAGE101 95
PAGE102 96
PAGE103 97
STRUCT2 other
ODIV1 Main
FILES1
FILES2