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Cellular mechanisms for the regulated degradation of aldolase B

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Cellular mechanisms for the regulated degradation of aldolase B
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CELLULAR MECHANISMS FOR
THE REGULATED DEGRADATION OF ALDOLASE B












By

Peter P. Susan


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


1998















TABLE OF CONTENTS

page

A B S T R A C T ........................................................................................................... vi

CHAPTERS

1 IN T R O D U C T IO N ............................................................................................... 1


General Concepts of Protein Degradation..................................................... 1

Background for Fructose 1,6-Diphosphate Aldolase B................................. 3

Mechanisms for Degradation Aldolase B...................................................... 8


Hypothesis for Stress-Induced Degradation ofAldolase B.......................... 28

G general Strategy........................................................................................ 30

2 MATERIALS AND METHODS........................... .......................................... 33


Cell Lines and Culturing............................................................................. 33


Plasmid Vector Construction and Mutagenesis........................................... 35











Expressing Epitope-Tagged Aldolase B in Cell Lines.................................. 41

Immunofluorescence.................................................................................. 44

Antibodies.................................................................................................. 44

Viability Assays.......................................................................................... 48

Subcellular Fractionation............................................................................ 49

Enzym e Assays.......................................................................................... 52

Protein Analysis......................................................................................... 53

Stress-Induction of Protein D egradation..................................................... 55


Protein Degradation................................................................................... 57

3 UBIQUITINATION MEDIATES LYSOSOMAL PROTEOLYSIS OF
ALD OLA SE B................................................................................................ 59
Introduction............................................................................................... 59

In Vivo M ultiubiquitination of Aldolase B................................................... 60

Multiubiquitinated Aldolase B is Denatured and Enriched in Lysosomes.....68

Heat Stress-Induced Delivery of Aldolase A to Lysosomes Requires
Ubiquitination......................................................................................... 69

Heat Stress-Induced Proteolysis of Aldolase B Requires Ubiquitination...... 75










Ubiquitin-Mediated Autophagic Degradation Occurs in E36AB Cells..........93

4 TEMPERATURE MODULATES AUTOPHAGY AND CYTOSOLIC
PROTEOLYSIS OF ALDOLASE B............................................................... 101
Introdu ctions............................................................................................. 10 1

Ubiquitin-Independent Cytosolic Proteolysis of Aldolase B....................... 102

Temperature-Dependent Cytosolic Proteolysis in Fao Cells........................ 106


Starvation-Induced Autophagic Degradation of Aldolase B in Fao Cells.... 110

Temperature-Dependent Autophagy and Cytosolic Proteolysis.................. 118

A Model For the Degradation of Aldolase B.............................................. 123

5 SIGNAL-MEDIATED DEGRADATION OF ALDOLASE B........................... 127

Intro du action ............................................................................................... 12 7

Transient Expression of RABM Mutations in Putative Lysosome
T argeting Signals.................................................................................... 130

Starvation Induces Autophagic Degradation in HuH7 Cells........................ 139

Transient Expression Does Not Affect Starvation-Induced Degradation
o f R A B M ............................................................................................... 14 0
Site-Directed Mutations Did Not Affect Wildtype Activity of RABM........142

Glutamine Residue #111 is Required for Starvation-Induced Degradation
of A ldolase B ......................................................................................... 143
Glutamine #111 Specifically Mediates Starvation-Induced Degradation of
A ldo lase B ................................................................................................. 14 6
6 SUMMARY AND CONCLUSIONS................................................................. 151


iv










Introduction............................................................................................. 151

Autophagy and Ubiquitination.................................................................. 151


Clues from Temperature-Dependent Cytosolic Proteolysis and
Lysosomal Degradation......................................................................... 154
Signal-M ediated Targeting....................................................................... 157

Present and Future Contributions to the Field of Protein
Turnover............................................................................................... 153
REFERENCES.................................................................................................... 164

BIOGRAPHICAL SKETCH ................................................................................ 176















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

CELLULAR MECHANISMS FOR
THE REGULATED DEGRADATION OF ALDOLASE B

By

Peter P. Susan

August 1998

Chairman: William A. Dunn, Jr.
Major Department: Anatomy and Cell Biology

Stress-induced degradation of abundant long-lived cytosolic housekeeping

proteins was examined using liver aldolase B as a model protein. Heat stress increases

ubiquitination that mediates autophagic degradation of long-lived proteins in E36

Chinese hamster cells. During starvation, major multiubiquitinated proteins (e.g., Ub68)

increased in lysosomes (rat liver and Fao hepatoma cells) and were antigenically

characterized as aldolase B-ubiquitin conjugates. Compared with controls, heat stress

increased endogenous aldolase A activity in lysosomes of E36 cells by >twofold. Heat

stress was non-permissive for ubiquitination in E36-derived ts20 mutant cells and failed

to increase aldolase activity in ts20 lysosomes. Myc-tagged aldolase B (RABM)

expressed in E36 cells underwent limited proteolytic processing in lysosomes that failed










to occur in heat stressed ts20 cells. The results suggested that during stress (starvation

or heat), aldolase A and aldolase B can undergo ubiquitin-mediated autophagic

degradation. Long-lived protein degradation was a continuous function of temperature,

indicating heat stress-induced rates were due to thermodynamic stimulation of chemical

reactivity. Lysosomal inhibitors distinguished proteolysis in lysosomes from that in

cytosol. Complete autophagic degradation to amino acids in lysosomes was highly

temperature-dependent compared to a relatively constant rate in cytosol. HA-tagged

human aldolase B (HAHAB) in Fao cells and RABM in E36 cells underwent proteolysis

in cytosol that had temperature-dependence paralleling complete degradation of proteins

in lysosomes. Lysosomal degradation was ubiquitin-dependent (blocked in heat stress

ts20 cells), but cytosolic proteolysis of RABM was not. Results suggest a possibly

shared temperature-dependent cytosolic mechanism that limits rates for partial cytosolic

proteolysis and complete lysosomal degradation of long-lived proteins. Three peptide

motifs for signal-mediated targeting to lysosomes during starvation occur in aldolase B.

These were mutated in RABM. Starvation-induced degradation of mutant and wildtype

RABM expressed in HuH7 human hepatoma cells were measured. Starvation-induced

degradation of RABM (aldolase B) specifically required a glutamine at residue #111

suggesting that the corresponding peptide motif, IKLDQ, is a targeting signal

functionally demonstrated in living cells. Evidence was provided for three previously

unknown mechanisms for stress-regulated degradation of aldolase B: (1) ubiquitin-










mediated autophagic degradation in lysosomes, (2) temperature-dependent cytosolic

proteolysis during heat stress, and (3) signal-mediated degradation during starvation.















CHAPTER 1:
INTRODUCTION

General Concepts of Protein Degradation

In cells, different proteins have different functions and occur at different levels as

needed. The functional activities and locations of proteins are regulated to integrate with

each other, maximizing survival. Proteins can be regulated by a variety of mechanisms,

but available concentration of each protein fundamentally determines maximal function

(Doherty and Mayer, 1992). Cells adapt to environmental change by altering amounts of

different proteins. Some increase, others decrease, and the rest are constant (Doherty

and Mayer, 1992). Such adaptation of different proteins requires preferential

mechanisms for regulating synthetic or degradative rates in response to environmental

change.

Under constant conditions, protein synthesis is independent (zero order

relationship) of the protein concentration, but degradation is directly proportional (first

order relationship) to protein concentration (Doherty and Mayer, 1992). Synthesis

increases protein concentration, causing degradation to increase until synthetic and

degradative rates are equal. In this way, a balance between synthesis and degradation

determines the available concentration of a protein. If environmental conditions change,

then cells can adapt protein concentrations by modulating synthesis or degradation










(Olson, et al., 1992; Mortimore, 1987). This study examines mechanisms of protein

degradation that respond to environmental changes.

Continual synthesis and degradation results in constant turnover of proteins

which can be described by either the fractional degradation rate or the half-life of the

protein. The fractional degradation rate of a protein (degradative rate constant), kd, is

defined as the fraction of the initial protein degraded in a given time. The kd is calculated

from measurements of labeled protein lost per time. Half-life, tv2, is defined as the time

for turnover of half the protein. Under equilibrium conditions, ka and ty, are constant and

directly related to each other by t./, = ln(2)/ kd, allowing calculation of t% from

experimentally determined kd (Doherty and Mayer, 1992).

Proteins are categorized as short-lived for ty < 1 h or long-lived for t% >1 h. For

short-lived proteins, protein concentrations respond more to changes in synthesis (Olsen,

et al., 1992). For long-lived proteins, protein concentrations respond more to changes in

degradation (Olsen, et al., 1992; Mortimore and Poso, 1987). Detailed reasoning for

this is described elsewhere (Doherty and Mayer, 1992). Many short-lived proteins utilize

a well-characterized mechanism for degradation by a cytosolic protease complex called

the proteasome (Rock, et al., 1994; Ciechanover and Schwartz, 1994; Hochstrasser,

1992). Relative to short-lived proteins, mechanisms for the degradation of long-lived

proteins are poorly characterized. This dissertation examines mechanisms for stress-

inducible degradation of a long-lived cytosolic enzyme, fructose 1,6-diphosphate

aldolase (see next section).










Mechanisms for degradation of proteins become enhanced during environmental

stress. Increased temperature (Bates, et al., 1982; Hough and Reichsteiner, 1984) or

nutrient deprivation (Mortimore and Poso, 1987) are known to increase the degradation

of long-lived proteins. Stress-induced degradative mechanisms are of special interest,

because they mediate regulated changes and themselves must be regulated (Mortimore,

et al., 1987; Mortimore and Poso, 1987; Olson, et al., 1990). Any mechanism that can

be triggered by environmental stress lends itself to experimental manipulation. Such a

mechanism can be modulated simply by changing experimental conditions. Furthermore,

mechanisms required for a stress-induced degradation can be shown to be specific by

lack of effect on basal mechanisms. For example, cells respond to starvation by

increasing the degradation of long-lived proteins. 3-Methyladenine is a drug that

specifically blocks the enhanced degradation without affecting basal degradation. 3-

Methyladenine is a potent inhibitor of autophagy, a mechanism for delivering proteins to

lysosomes for degradation. Such results provide evidence that autophagy plays a role in

enhanced degradation but not basal degradation of proteins.

Background for Fructose 1 .6-Diphosphate Aldolase B

In the next section, I discuss potential mechanisms for the degradation of the

liver isoform of fructose 1,6-diphosphate (FDP) aldolase, called aldolase B. Molecular

mechanisms for the degradation of aldolase B have not been examined, but preliminary

examination indicated potential roles for ubiquitination and signal-mediated delivery to

lysosomes. Before examining the degradation of aldolase B, this section serves to










familiarize the reader with aldolase B in relation to other aldolase isoforms. There are

two major classes (I and II) ofFDP aldolase that have no sequence homology

(Alefounder et al., 1989) and utilize different catalytic mechanisms (Rutter et al., 1966).

Class II aldolases are only found in microorganisms and are considered no further here,

but class I aldolases are represented in all taxons (Rutter et al., 1966). Microorganisms

variably lack class I aldolase. For example, some strains of E.coli contain class I

aldolase (Alefounder et al., 1989), whereas strain JM83 lacks class I activity (Sakakibara

et al., 1989). Protozoa and muticellular eukaryotes contain class I aldolases.

The FDP aldolase isoforms of mammals are the best characterized. Most studies

ofFDP aldolase degradation examine mammalian isoforms. Mammalian aldolase

isoforms are synthesized from three separately regulated genes coding different proteins.

Muscle, liver, and brain each express one predominant isoform designated A, B, and C,

respectively (Rutter et al., 1966). As such, liver aldolase and aldolase B are synonyms.

Likewise, aldolase A is also called muscle aldolase, and aldolase C is referred to as brain

aldolase. Most tissues, including embryonic, contain combinations of aldolase A and C,

but aldolase B appears to be exclusively expressed in liver and kidney cells (Rutter et al.,

1966).

Aldolase classes and isoforms are distinguished by clear differences in their

enzymatic properties. For example, class I aldolase like that in mammals is totally

functional in the presence of EDTA, but class II enzymes ofE. coli and other microbes

are completely inhibited. The three mammalian isoforms (A, B, and C) can be










distinguished by differences in specific activity, sensitivity to carboxypeptidase A, and

kinetics (Vx and Kin) for different substrates (Rutter et al., 1966).

Aldolases A, B, and C also are characterized by distinct native epitopes. Thus,

antibody to one poorly recognizes the others. However, an antibody against a specific

isoform for one animal will similarly recognize the same isoform of a different species, at

least amongst mammals (Penhoet and Rutter, 1975). Native and denatured epitopes of

aldolase B have also been shown to be distinct from each other (Reznick et al., 1985).

Chemical denaturation of aldolase B before immunization resulted in antibody that failed

to immunoprecipitate native enzyme activity but could precipitate degradative fragments

of aldolase B. Since aldolase has stable structure that spontaneously refolds into native

conformations, only fragments sufficiently denatured by degradation were detected by

antibody against the denatured aldolase B (Reznick et al., 1985). Anti-native aldolase B

had converse immunoreactivity. Thus, it was proposed that three-dimensional

conformation is important for antibody recognition of native surface epitopes, whereas

the native structure buries and masks denatured epitopes (Reznick et al., 1985).

The three dimensional structure of all class I aldolase isoforms is conserved from

bacteria to humans (Alefounder, et al., 1989). Secondary and tertiary structures of

aldolase are very stable. This is true at the quaternary level, too. Aldolase occurs as

very stable tetramers that do not undergo subunit exchange after synthesis (Lebherz,

1975; Lebherz, 1972). Different aldolase isoforms co-synthesized in the same cell

randomly associate into stable heterotetramers. Thus, immunoprecipitation with










antibody against one isoform specifically precipitates antigenically unrelated isoforms in

the same tetramer. However, surface charge and pI on different isoforms varies. Thus,

isozymes containing different ratios of two isoforms (e.g. A4, A3B, A2B2, AB3, or B4)

can be separated by isoelectric focusing (Lebherz, 1972; Penhoet and Rutter, 1975).

Comparison of X-ray crystallographic results shows that secondary and tertiary structure

between muscle, liver, and Drosophila aldolases are very close (Berthiaume et al., 1993).

Aldolase isoforms vary in their capacity to bind actin cytoskeletons. In the only

paper to measure cytoskeletal association of all three isoforms (A, B, and C),

investigators claimed that different purified isoforms of aldolase had tissue-specific

affinity for cytoskeletal preparations isolated from different tissues (Kusakabe et al.,

1997). After mixing crude cytoskeletons with a known amount of purified aldolase, they

pelleted the mixture and then only measured unbound activities in supernatants. They

failed to show removal of endogenous aldolase from cytoskeletal preparations, so

measurements could be contaminated and include competitive effects. However, their

results were consistent with other investigators in that relative tightness of binding to

actin cytoskeleton is greatest to least, aldolase A, aldolase B, then aldolase C (Clarke, et

al., 1982; O'Reilly and Clarke, 1993).

Four "isotype specific" sequences contain most of the variation and the carboxyl

terminus has the greatest diversity (Marchand et al., 1988; Paolella et al., 1984;

Rottmann et al., 1984). The carboxyl terminus is important in determining isoform-

specific catalytic properties (Berthiaume et al., 1993; Gamblin et al., 1991; Penhoet and









7
Rutter, 1975). Aldolase B has the lowest specific activity amongst aldolase isoforms and

is least sensitive to proteolytic alterations in this region. It can lose up to four C-terminal

residues without affect its enzymatic activity (Berthiaume et al., 1993; Horecker et al.,

1985). Even with 10 to 20 residues removed by carboxypeptidase, aldolase B retains

almost half its activity (Penhoet and Rutter, 1975). However, aldolase A absolutely

requires a carboxyl terminal tyrosine at residue 364 (Y364) for activity that is about 20

times greater than aldolase B, and when aldolase A loses its C-terminus the remaining

activity resembles that of aldolase B (Takahashi et al., 1989; Gamblin et al., 1991).

These results indicate that alterations in the carboxyl terminus of aldolase B (such as

epitope tagging) are less likely to affect its properties than other aldolase isoforms.

All fructose 1,6-diphosphate aldolase enzymes catalyze a reversible reaction

essential for glycolysis and gluconeogenesis. Aldolase B is the liver form of this enzyme

expressed to the exclusion of other forms of aldolase in normal hepatocytes (Asaka, et

al., 1983). Since liver is the only organ known to export glucose (Stein and Arias, 1976;

Stryer, 1988), aldolase B performs gluconeogenesis for the entire body. Liver also

provides amino acids during starvation and in three days can lose nearly half its weight

(and protein content), a faster loss than other tissues (Wing et al., 1991). In this regard,

aldolase B is an example of an abundant cytosolic protein that undergoes enhanced

degradation during starvation which yields amino acids for export to other organs. Liver

amino acids can also be converted to glucose or ketone bodies to provide energy sources

during starvation. Abundant long-lived liver enzymes that mediate












glycolysis/gluconeogenesis, like aldolase B and glyceraldehyde phosphate dehydrogenase

(GAPDH) are poised between two mutually exclusive functions: catalyzing carbohydrate

metabolism and providing amino acids for protein biosynthesis or energy metabolism.

Liver and kidney are the only tissues having predominant aldolase B expression

(Penhoet and Rutter, 1975). Both organs demonstrate enhanced degradation of proteins

during amino acid starvation (Olsen, et al. 1990). Liver and kidney also receive the

largest fraction of the body's basal blood flow, 27% and 22%, respectively, followed by

15% for muscle and 14% for brain (Guyton, 1979). This is consistent with involvement

of the two former organs in regulating serum components and contribution of aldolase B

to serum glucose and amino acids during starvation. Among aldolase isoforms, aldolase

B contributes a greater role in carbohydrate and protein metabolism that is not limited to

local cells and tissues, but extends to the entire body.

Mechanisms for Degradation Aldolase B

Inactivation by Limited Proteolysis

Alteration of aldolase A and B's carboxyl termini was proposed to down-regulate

activity (Pontremoli et al., 1982; Pontremoli et al., 1979). During starvation, aldolase B

activity is lost from liver faster than loss of immunoreactivity. Thus, investigators

suggested that starvation-induced inactivations precede total degradation of aldolase A

and B, providing more rapid down-regulation of activity (Pontremoli et al., 1979).

Inactivation happens by limited C-terminal cleavage that can maintain native








9
immunoreactivity and barely affect mobility on SDS-PAGE. One group of investigators

proposed phosphorylation near the C-terminus of aldolase as an inactivating mechanism,

but this was only demonstrated in vitro (Sygusch et al., 1990). More likely inactivation

occurs by limited proteolysis which would have a much more profound impact on

aldolase A activity than on aldolase B activity (previous section, last paragraph).

The best characterized mechanism for aldolase inactivation is limited proteolysis

by a dipeptidyl (two residues per cleavage) carboxypeptidase on lysosomes (Pontremoli

and Melloni, 1986; Horecker et al., 1985). The peptidase, cathepsin M, was defined as

a cathepsin B or L-like activity associated with the cytosolic surface of lysosomal

membranes. During starvation, a lysosomal matrix cathepsin B/L associates with

lysosomal membranes, acquires activity at neutral pH, and becomes exposed to the

cytosolic compartment as cathepsin M (Pontremoli et al., 1984; Pontremoli et al., 1982).

Specific cleavage sites have been characterized in vitro (Horecker et al., 1985).

Starvation-induced in vivo loss of liver aldolase specific activity correlated with

loss of carboxyl terminal tyrosine residues which was estimated by isolating aldolase B

and measuring lost tyrosine content in an acid soluble peptide released from the C-

terminus with subtilisin (Pontremoli et al., 1982). According to such experiments,

inactivated aldolase B constitutes about 40% of the aldolase in liver after 60 hours of

starvation. Most of the inactivated aldolase B must occur in cytosol, because only a

small fraction of total aldolase (about 10%) is associated with pelletable fractions from

liver (Kominami et al., 1983; Kopitz et al., 1990). Moreover, intralysosomal










degradation of aldolase is rapid (see below), precluding accumulation of an inactivated

form in such organelles. The results are consistent with inactivation occurring in the

cytosolic compartment, albeit by an activity associated with the cytosolic surface of

lysosomes.

In Vitro Denaturation of Aldolase and Need for In Vivo Mechanism

Except for 20 "loose" amino acid residues at the carboxyl terminus, the stability

of aldolase structure resists proteolysis and requires denaturation for rapid in vitro

proteolysis to proceed. In optimized conditions with cathepsin D, only about 20 amino

acids of aldolase A can be digested from its carboxyl terminus (Offermann et al., 1983).

In vitro proteolysis with either meprin (a metalloproteinase) or a mixture of lysosomal

proteases produces only a slight increase in SDS-PAGE mobility, and the remaining part

of aldolase A has a thermal stability identical to the native enzyme (Bond and Offermann,

1981). Purified aldolase B digested with a lysosomal extract also only undergoes

limited proteolysis, losing some but not all its activity (Chappel et al., 1978). However,

denaturing pretreatment with disulfides like glutathione (Offermann et al., 1983) or

cystine (Bond and Offermann, 1981) permits extensive proteolysis to occur. Given

this, there must be a denaturingg" mechanism in vivo to allow degradative turnover of

aldolase to occur. Interestingly, aldolase B sequestered in vivo and isolated with

lysosomes is susceptible to more extensive in vitro proteolysis in the lysosomal

preparations (Kominami, et al., 1983; Ueno and Kominami, 1991). Apparently,










aldolase B becomes sensitized to proteolysis by a mechanism in cytosol before

sequestration or in intact lysosomes after sequestration.

After loading aldolase A into endosomes at 19C, temperature can be raised to

37C allowing rapid fusion of endosomes with lysosomes. Thus, intralysosomal

degradation can be measured. By this method, native or variously denatured and

inactivated aldolases all degrade rapidly with similar rates (t, < 10 min). Since its t, is

normally many hours in cytosol, sequestration appears rate limiting for lysosomal

degradation of aldolase (Bond and Aronson, 1983). The results of the endocytic loading

experiments indicate that a mechanism for denaturing and sensitizing aldolase to

proteolytic attack can occur in lysosomes or other organelles of the endosomal pathway.

Thus, a cytosolic denaturing mechanism is not necessary for intralysosomal degradation

of aldolase, but a role in delivery of aldolase to lysosomes cannot be excluded.

The tetramrneric structure of aldolase is well established (Lebherz, 1972). This

quaterenary structure seems important for aldolase stability. Recently, Beemrnink and

Tolan have identified specific amino acids that mediate subunit interaction between

aldolase monomers (Beemrnink and Tolan, 1996). Significantly, a mutant with only two

amino acid changes retains enzymatic activity but exists as monomers. These monomers

(and dimers created with single amino acid mutations) are more sensitive to chemical or

thermal inactivation, indicating "looser" structure. Thus, tetrameric association

improves structural stability.










Lysosomes are acidic inside (pH -5), and reversible in vitro dissociation of

aldolase into monomers occurs at pH < 6.0 (Beernink and Tolan, 1996). Acidic pH

affecting adolase structure is also indicated by reduced enzymatic activity. Thus,

intralysosomal pH would have a denaturing effect that could permit lysosomal

proteolysis. However, other investigators incubated aldolase B with crude lysosomal

hydrolases at acidic pH and failed to get significant proteolysis (Chappel et al., 1978).

Apparently, low pH is insufficient to permit further proteolytic attack, and aldolase

denaturation must require other factors. Consistent with this, lysosomes purified from

liver contain detectable aldolase B which is susceptible to proteolysis when the intact

lysosomes are incubated in vitro at pH < 5.5 (Kominami, et al., 1983; Ueno and

Kominami, 1991). The endocytic loading experiments described above indicate

lysosomes (or an endocytic compartment) must contain denaturing factors, but this does

not exclude the possibility of a cytosolic denaturation of aldolase B before delivery to

lysosomes.

Aldolase A has been radiolabeled, inactivated and denatured, then microinjected

into cultured cells (Hopgood et al., 1988; Knowles et al., 1989). The procedure delivers

the enzyme into cytosol where it normally resides. As with endocytic loading,

degradation rates for aldolase were similar whether the enzyme was native, inactivated,

or denatured. Denaturation of aldolase is not rate limiting for degradative steps before

lysosomes as well as within them. Degradation of aldolase microinjected into cytosol

matched expected turnover for aldolase (ty = 30 hours) which was much slower than for










aldolase loaded into lysosomes (ty, < 10 minutes). Assuming that degradation occurs

within lysosomes, this suggest that sequestration of aldolase is rate-limiting for its

turnover (Bond and Aronson, 1983; Bond and Offermann, 1981; Hopgood et al., 1988;

Knowles et al., 1989).

Though in vitro studies indicate denaturation of aldolase structure is necessary

for proteolysis, in vivo denaturation is not rate-limiting for delivery to or degradation

within lysosomes. These data support a model in which aldolase delivery to lysosomes is

rate limiting followed by rapid intralysosomal proteolysis which would need a faster

denaturing mechanism. Lysosomal acidity might facilitate denaturation of aldolase, but

acidity alone is insufficient for sensitizing stable aldolase structure to attack by acid

hydrolases. The above data do not exclude a cytosolic denaturing mechanism for

aldolase, but indicate that such a mechanism is not rate limiting and not necessary for

intralysosomal proteolysis. The next two sections review mechanisms for the delivery of

cytosolic proteins to the lysosomal lumen, a process that appears rate-limiting for

aldolase degradation.

Autophagy

Autophagy is the sequestration of cytoplasm into vesicles for intralysosomal

degradation and is the only mechanism proposed for the complete degradative turnover

of aldolase. There are two forms of autophagy: macroautophagy and microautophagy.

Commonly, investigators use the term "autophagy" to mean macroautophagy which is










the better characterized form. Likewise, "autophagy" used here refers to

macroautophagy, and reference to "microautophagy" will be explicit.

Autophagy (macroautophagy) begins with a ribosome-free portion of

endoplasmic reticulum engulfing a portion of cytoplasm. Autophagy non-selectively

sequesters cytosol and organelles into distinct autophagic vacuoles. The autophagic

vacuoles mature including a process of acidification. Finally, mature autophagic

vacuoles fuse with lysosomes producing autolysosomes in which degradation occurs

(Dunn, 1990; Dunn, 1990). Enhanced autophagy is initiated by amino acid starvation

and is also regulated by hormones (Hendil et al., 1990; Seglen and Bohley, 1992). In the

model of Figure 1-1, non-selective autophagy is represented by the upper pathway in the

diagram. The lower pathway of Figure 1-1 (Receptor-Mediated Targeting) is discussed

in the next section.

Microautophagy seems simpler than macroautophagy. During microautophagy

the lysosomal membrane itself invaginates, extending a finger of cytosol into the

lysosome. This protrusion pinches off producing an intralysosomal vesicle that gets

degraded with its cytosolic content. Apparently, microautophagy can occur in vitro, but

the complexity of macroautophagy has not been reconstitiuted (Seglen and Bohley,

1992). Unlike macroautophagy which has discrete autophagic vacuoles, microautophagy

fails to produce separable organellar compartments. Thus, microautophagy requires

time-consuming electron microscopy to demonstrate its existence and remains poorly

characterized (Seglen and Bohley, 1992).

































Figure 1-1: Mechanisms for Stress-Induced Degradation of Cytosolic Proteins in
Lysosomes. Autophagy (upper pathway) and receptor-mediated targeting (lower
pathway) were proposed for stress-induced delivery ofcytosolic proteins to lysosomes
for degradation; the arbitrary cytosolic protein is shown as a tetramer (aldolase B occurs
as a tetramer); components of the pathways are labeled on the diagram; processes are
labeled by boxed numbers: 1, association with or engulfminent by autophagic membranes;
2, sequestration into double-membrane bound autophagic vacuole; 3, maturation of
autophagic vacuole (acidification and acquisition of lysosomal hydrolases); 4, proteolysis
into polypeptide fragments; 5, complete degradation to amino acids; 6, disassembly and
denaturation of structure by an unknown factor; 7, association with a receptor complex
on the lysosomal surface; 8, translocation across the lysosomal membrane.



Selective mechanisms of autophagy exist. Methylotrophic yeast use a selective

mechanism of autophagy to degrade peroxisomes when switched from methanol to a

different carbon source, and electron microscopic morphology shows a mechanism









16
topologically identical to microautophagy (Tuttle et al., 1993). Occurrence of selective

microautophagy in higher organisms has not been demonstrated, and a role for

microautophagy in degradation of aldolase has not been studied. If selective autophagy

does occur for aldolase, then a receptor-mediated complex would be required for

selectivity. Such a receptor complex could form on the lysosomal surface (Fig. 1-1,

lower pathway), followed by microautophagic sequestration. However, receptor

function does not distinguish microautophagy and macroautophagy, so Figure 1-1 only

distinguishes non-selective autophagy (upper pathway) from a hypothetical selective

process that might include microautophagy (lower pathway).

Autophagy (macroautophagy) is a subject of active research, producing almost

300 related papers in just the last five years. FDP aldolases are generally abundant,

commonly known, cytosolic enzyme, and the muscle isoform, aldolase A, is

commercially available. Aldolase A and aldolase B have been used as markers for

autophagic uptake of cytosol into lysosomes, and degradation of aldolase by autophagy

is well established (Henell et al., 1987; Kominami et al., 1983; Kopitz et al., 1990;

Seglen and Gordon, 1982; Ueno et al., 1990). Per 0. Seglen's laboratory briefly treated

starved hepatocytes with cycloheximide to prevent new protein synthesis and estimated

degradation rates by loss of enzyme activity. Incubations were short to avoid depletion

of autophagic factors (continual autophagy requires new protein synthesis). A lysosomal

inhibitor (leupeptin) was used to estimate how much degradation occurred in lysosomes.

In this way, starvation-induced degradation of aldolase B was lysosomal occurring at










3.60.1 %/h. Other cytosolic enzymes with widely different half-lives were similarly

tested. As expected, they had very different total degradation rates. However,

lysosomal degradation (3.3-5.3 %/h) and rates of accumulation in organelles during

lysosomal inhibition (3.1-3.7 %/h) were similar for all the enzymes. These rates match

rates of starvation-induced autophagy (3-5 %/h) and were blocked with 3-methyladenine

a specific inhibitor of autophagy. Thus, Seglen concluded that cytosolic enzymes,

including aldolase B, are degraded via non-specific autophagy (Kopitz et al., 1990).

Interestingly, of all the enzymes tested by Seglen, only two were exclusively degraded in

lysosomes, aldolase B and lactate dehydrogenase H (Kopitz et al., 1990).

Coincidentally, both aldolase B and lactate dehydrogenase H contain sequence motifs for

receptor-mediated targeting to lysosomes for degradation.

Receptor Mediated Targeting to Lysosomes

A pentapeptide sequence (KFERQ) of RNAse A was shown to mediate its

delivery to lysosomes for degradation during nutrient deprivation (Dice and Chiang,

1988). Characterization of this signal identified a motif contained in a subset of cellular

proteins that undergo enhanced degradation in lysosomes during nutrient deprivation

(Wing et al., 1991). The motif has been proposed as a binding site for a molecular

chaperone called HSC73 which then delivers motif-containing proteins into lysosomes in

an ATP-dependent manner (Chiang et al., 1989). The mechanism (Figure 1-1, lower

pathway) also requires intralysosomal HSP70, and a recently identified lysosomal

membrane receptor, LGP96 (Cuervo, et al. 1996). J. Fred Dice proposed that this










pathway occurs by a mechanism analogous to transmembrane transport of proteins

during organellar biogenesis (Dice and Chiang, 1988; Terlecky et al., 1992; Wing et al.,

1991).

Recently, Dice's group made a major advance by identifying a receptor protein in

the target membrane of lysosomes that mediated transmembrane translocation (Cuervo

and Dice, 1996). The lysosomal membrane protein, LGP96, was demonstrated to be a

rate limiting component in this degradative pathway. CHO cells overexpressing human

LGP96 by two to three fold had correspondingly increased degradation of long-lived

proteins. Furthermore, lysosomes isolated from these cells had two to three fold higher

ATP-dependent uptake of the glycolytic enzyme GAPDH (glyceraldehyde 3-phosphate

dehydrogenase), a known substrate for his pathway (Cuervo and Dice, 1996).

Unfortunately, GAPDH does not contain sequence matching previously established

criteria for the receptor-mediated targeting motif The previous criteria define necessity

for an "essential" glutamine, but in GAPDH, asparagine apparently can substitute for

glutamine (Dice, personal communication). As just described, receptor function has been

demonstrated in living cells; however, evidence for in vivo function of the signal peptide

is lacking.

A recent study found that the conformation of signal motifs were inappropriate to

mediate the receptor-mediated lysosomal targeting pathway (Gorinsky et al., 1996).

Some proteins known to contain motifs for the pathway, including RNAse A, also have

known three dimensional structures. The peptide signal motifs are supposed to be










recognized by cytosolic HSC73 which is required for delivery to lysosomes. However,

signals on proteins of known structure are either embedded or occurred in a-helical

conformations. Since hsp70-type chaperones require extended conformations for

recognition, the investigators concluded that HSC73 would required other unknown

factors to relax a substrate protein's structure and allow signal-mediated targeting to

lysosomes to occur (Gorinsky et al., 1996). The lower pathway in Figure 1-

1 summarizes receptor-mediated targeting to lysosomes, including an unknown factor

that alters the structure of the cytosolic protein.

Except for the work presented in this dissertation, no studies have examined any

aldolase isoform as a substrate for the receptor-mediated pathway. Aldolase A binds

very well to GAPDH presumably for greater glycolytic efficiency (Verlessy and Vas,

1992). Both these proteins appear to be regulated at similar high concentrations in cells

(Verlessy and Vas, 1992), and aldolase B and GAPDH undergo similar starvation-

induced degradation in cultured cells (Kopitz, et al., 1990). Does aldolase B follow

receptor-mediated targeting to lysosomes like GAPDH?

All vertebrate aldolases contain a conserved motif (Fig. 1-2, residues 12-16) for

receptor-mediated targeting of cytosolic proteins into lysosomes (Dice and Chiang,

1988; Zhang et al., 1995). In mammalian aldolase B, two additional sequences for the

lysosomal targeting motif were found (Fig. 1-2, residues 58-62 and 107-111). Whether

any of these three motifs are functional was unknown. Though the lysosomal targeting

motif has rather broad criteria (Dice and Chiang, 1988), an aldolase-sized (40 kD)














MAHRFPALT S EQKKELSEI AQRIVANGKGILAADESVGTMGNR
MAHRFPALTQEQKKELSEI AQRIVANGKGILAADESVGTMGNR
18

LQRIKVENTEENRRBFRELLFSVDNSISQSIGGVILFHETLYQKDS
LQRIKVENTEENRRQFRE I LFSVDNSISQSIGGVILFHETLYQKDS
44 Qlll

QGKLFRNILKEKGIVVGIKLD GGAPLAGTNKETrIQGLDGLSER
QGKLFRNILKEKGIVVGIKLDOGGAPLAGTNKETTIQGLDGLSER
90

CAQYKKDGVDFGKWRAVLRIS DQCPSSLAIQENANALARYASIC
CAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASIC

135QQNGLVPIVEPEVLPDGDHDLEHCQYVSEKVLAAVYKALNDHH


QQNGLVPIVEPEV I PDGDHDLEHCQYVT SEKVLAAVYKALNDHH
QQNGLVPIVEPEV I PDGDI-DLEHCQYVTEKVLAAVYKALNDHH
179
VYLEGTLLKPNMLTAGHACTKKYTPEQVAMATVTALHRTVPAA
VYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAA
221
VP S ICFLSGGMSEEDATLNLNAIYRCPLPRPWKLSFSYGR ALQAS
VPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGKALQAS
265

ALAAWGGKAANKKATQEAFMKRAV ANCQGQYVHTGSSGAAS
ALAAWGGKAANKEATQEAFMKRAMANCKGQYVHTGSSGAAS
300

TQSLFTA SYTY
TQSLFTACYTY
354 364

Figure 1-2: Amino Acid Sequences ofAldolase B Isoforms Used in This Study. The
upper and lower sequences are for rat and human liver aldolase, respectively. Boldface
indicates non-identical residues. Underline indicates pentapeptide signal motifs for
receptor-mediated lysosomal targeting. Large arrows point to essential glutamines of the
signals as indicated (sequence data from Tsutsumi et al., 1984 and Paolella et al., 1984).









protein would have only a 7% chance of randomly containing three such signal motifs.

In addition to the motifs, aldolase B has properties similar to other substrates of this

signal-mediated degradative mechanism: (1) long-lived, (2) cytosolic, (3) housekeeping

protein, and (4) degraded in lysosomes by enhanced proteolysis during nutrient

withdrawal. Furthermore, aldolase is closely associated with another glycolytic enzyme

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is an established substrate

for receptor-mediated targeting to lysosomes (Aniento et al., 1993). Aldolase and

GAPDH form a complex that facilitates their sequential roles in glycolysis (Verlessy and

Vas, 1992), both are very abundant, and their in vivo turnover rates are very similar

(Kuehl and Sumsion, 1970), suggesting that they could share degradative mechanisms.

Furthermore, the receptor-mediated pathway proceeds by transmembrane translocation

into lysosomes by a mechanism like that oforganellar biogenesis. Coincidentally, the

aldolase isoform of Trypanosoma brucei (45% identical to aldolase B) undergoes

transmembrane transport during biogenesis of the unique glycolytic organelle

of this protozoan (Marchand et al., 1988). Together, these facts suggested that aldolase

B was a likely candidate for receptor-mediated targeting to lysosomes via the proposed

transmembrane transport mechanism.

Ubiquitination and the Degradation of Long-lived Proteins

Ubiquitination is an orderly process whereby a 76-amino acid polypeptide,

ubiquitin, is covalently conjugated to other proteins at its carboxyl terminus. In a series

of transfers, three enzymes (El, E2, and E3) covalently bind and pass ubiquitin to the









22
next protein. El called ubiquitin-activating enzyme, first conjugates ubiquitin's carboxyl

terminus. This step is obligatory, and cell lines with temperature-sensitive ubiquitination

have defects traced to mutations in El (Kulka, et al., 1988; Chowdary, et al., 1994). El

transfers ubiquitin to an E2 which transfers it to an E3 which finally conjugates the

ubiquitin to a target protein (sometimes, the E3 step is skipped). Most protein

ubiquitination requires a single El protein, but E2 and E3 enzymes occur as families that

regulate and confer specificity for ubiquitination. This arrangement explains why genetic

defects in general ubiquitination only occur in El enzymes (Ciechanover and Schwartz,

1994; Hochstrasser, 1992).

Cells die without ubiquitination. The process has been implicated in a wide

variety of cell functions reviewed elsewhere (Hochstrasser, 1996; Jentsch, 1992).

Ubiquitination was originally discovered in the rapid degradation of short-lived and

abnormal proteins by cytosolic proteases, and this role remains the best characterized

(Hershko and Ciechanover, 1992). Heat stress causes enhanced degradation of long-

lived proteins in E36 Chinese hamster lung cells. This heat-stress induced degradation

occurs in lysosomes via autophagy and requires ubiquitin-activating enzyme El (Gropper

et al., 1991; Handley-Gearhart et al., 1994). However, specific long-lived proteins that

utilize this ubiquitin-mediated autophagic mechanism have not been identified.

Whether for short-lived or long-lived proteins, ubiquitin-mediated turnover

involves attachment of multiple ubiquitins on a protein targeted for degradation

(Hershko and Ciechanover, 1992). A ubiquitinated protein (a.k.a. ubiquitin conjugate) is









23
a substrate for further ubiquitination, and additional ubiquitins preferentially conjugate to

already attached ubiquitin. A chain of ubiquitins is built on a protein to be targeted. The

multiubiquitin chain then acts as a signal for degradation of the targeted protein. Each

ubiquitin adds an additional 76 amino acids to the protein, and successive intermediates

of multiubiquitination can be demonstrated as a ladder of bands on SDS-PAGE that

contain both ubiquitin and the targeted protein (Chau et al., 1989; Hershko and

Ciechanover, 1992). Multiubiquitination is a well-established signal for stress-induced

degradation of short-lived proteins by a major protease complex in cytosol. Though

total long-lived proteins undergo ubiquitin-mediated stress-induced degradation in

lysosomes, a role for multiubiquitination has not been established for lysosomal

degradation of any specific cytosolic protein.

Recently, work in the laboratory of William A. Dunn, Jr. demonstrated a

connection between ubiquitin and the long-lived protein aldolase B. The evidence

includes data presented in this dissertation, two meeting abstracts, and a manuscript

which has been submitted (Lenk et al., Submitted 1998; Susan and Dunn, 1996; Susan et

al., 1995). Together the data support that aldolase B is multiubiquitinated in vivo and

suggest that ubiquitination is involved with stress-induced autophagic degradation of

aldolase B in lysosomes.

S. E. Lenk and William A. Dunn, Jr. provided the first evidence that aldolase B

has ubiquitinated forms (Figs. 1-3 and 1-4), including a major 68 kD form (Ub68)

enriched in lysosomes during nutrient deprivation (Lenk et al., Submitted 1998; Susan et
























Figure 1-3: Characterization of A Major Ubiquitin-Protein Conjugate Enriched in
Autophagic Vacuoles. a) Rats were starved to induce autophagy and lysosomal uptake
of ubiquitinated proteins. Subcellular fractions of liver were prepared, and equal protein
from cytosolic (Cy), lysosome-enriched (Ly), and autophagic vacuole-enriched (AV)
fractions were run on SDS-PAGE, western blotted, and labeled with antibody against a
major ubiquitinated protein (anti-Ub68). Note major bands at 68 kD and cross-reactivity
to a 40 kD protein in cytosol. The 40 kD protein was identified as aldolase B by peptide
sequence analysis, b) Cytosol was circulated on an anti-Ub68 column, washed, eluted,
and preparative SDS-PAGE performed. A gel strip was stained with Coomassie blue R-
250 (CB), and the remaining gel was blotted and cut into strips individually stained with
anti-Ub68 (Ub68) or anti-ubiquitin (Ub). Ub68 and aldolase are indicated at 68 kD and
40 kD, respectively. Arrowheads indicate positions of bands suggestive of intermediates
of multiubiquitination of aldolase (from Lenk, et al., Submitted 1998).







a. Cy Ly AV b.
r.v




68 100> -, *- Ub6i

40
40 D,'Wu


Aldolase


Ub68 Ub


I|


-9
^f


F


anti-Ub68


3 sommii^^^ ^^^^


























Figure 1-4: Amino Acid Starvation Increases Lysosomal Association of Putative
Ubiquitinated Aldolase B via Autophagy. Fao rat hepatoma cells were incubated on
media with or without amino acids (AA) and the autophagy inhibitor 3-methyladenine
(3MA) as indicated. Sub-cellular fractions were collected, and equal protein of
lysosome-enriched fractions were run on SDS-PAGE, western blotted, and stained with
antibodies against Ub68 and ubiquitin (Ub). Positions of molecular weight standards and
Ub68 (arrowhead) are indicated (from Lenk et al., Submitted 1998).










Anti-Ub68


205-



116-

97-



66-


45-


29-


AA
3MA


+


Anti-Ub









28
al., 1995). Aldolase A and B are long-lived proteins known to undergo degradation by

autophagy (Henell et al., 1987; Kominami et al., 1983; Kuehl and Sumsion, 1970; Ueno

and Kominami, 1991; Ueno et al., 1990). It has been determined that amino acid

deprivation (starvation) rapidly enhances autophagy in cultured cells (Kopitz et al., 1990;

Seglen and Gordon, 1982; Ueno et al., 1990). Since Ub68 increases in lysosomes under

similar conditions (Fig. 1-4), the evidence suggested that ubiquitination might play a role

in stress-induced autophagic degradation of aldolase B.

Hypothesis for Stress-Induced Degradation of Aldolase B

The field of protein degradation has made great progress in determining

molecular mechanisms for the degradation of short-lived proteins via a cytosolic protease

complex (the proteasome); however, long-lived proteins which are generally thought to

be degraded in lysosomes have relatively poorly characterized degradative mechanisms.

Cellular degradative mechanisms that respond to environmental changes facilitate

experimental characterization. Starvation (amino acid and serum deprivation) and heat

stress can induce regulated mechanisms for the degradation of long-lived proteins.

Figure 1-1 presents two pathways proposed for the stress-induced delivery of cytosolic

proteins to lysosomes for degradation: autophagy or receptor-mediated targeting (lower

pathway).

In the simplified diagram of Figure 1-1, only topological changes in autophagy

(upper pathway) are shown for a tetrameric cytosolic protein sequestered into an

autophagic vacuole (steps 1 and 2) that fuses with lysosomes (step 3). Heat stress









29
induces autophagic degradation that requires ubiquitination, but specific proteins that are

ubiquitinated during stress-induced autophagy have not been identified. Aldolase B is

known to undergo autophagy, and a putative ubiquitinated form aldolase B associates

with autophagic vacuoles and lysosomes during starvation. To establish a specific

protein for ubiquitin-mediated autophagy, we examined aldolase B as a likely substrate

for ubiquitin-mediated autophagy.

In Figure 1-1, receptor-mediated targeting to lysosomes is also drawn showing

required components (lower pathway). Since established substrates for this pathway

have conformations that would prevent receptor recognition, unknown factors (smallest

circles) have been proposed to relax the structure of substrate proteins (first arrow)

which probably includes disassembly of subunits from quaternary structures (rough-

drawn oval with small circles attached). An exposed signal then mediates assembly of a

complex on the lysosomal surface (second arrow), including the HSC73 chaperone

(medium gray square), the substrate protein (extended coils), the lysosomal membrane

protein LGP96 (darkest rectangle), and possibly other factors (small circles).

Transmembrane translocation (third arrow) also requires an intralysosomal HSP70

chaperone (dark gray square). Aldolase B has characteristics similar to known substrates

for receptor-mediated targeting to lysosomes, but this mechanism was not examined for

any aldolase. Evidence will be shown that ubiquitinated forms of aldolase B have a more

denatured conformation. If aldolase B follows receptor-mediated degradation, then

ubiquitin could represent the unknown factor needed to relax substrate structure.










The relationship between receptor-mediated targeting of cytosolic proteins to

lysosomes and ubiquitin-mediated autophagic degradation had not been examined. Since

aldolase B was a potential substrate for both pathways, I hypothesized that during stress,

aldolase B requires both ubiquitination and a receptor-mediated targeting signal for

enhanced degradation in lysosomes.

General Strategy

I adopted the hypothesis that during stress, aldolase B requires both

ubiquitination and a receptor-mediated targeting signal for enhanced degradation. The

two requirements in this hypothesis were separately tested: ubiquitination and a receptor-

mediated targeting signal. In this regard, there were two corresponding aims of this

investigation: Aim #1, perturb ubiquitination and examine the effects on stress-induced

delivery of aldolase B to lysosomes; Aim #2, mutate potential lysosomal targeting signals

and examine effects on starvation-induced degradation of aldolase B.

My first aim was to determine whether stress-induced degradation of aldolase B

requires ubiquitination. Antibodies were raised against aldolase B expressed in and

isolated from E. coli. Since bacteria lack ubiquitin, the antibodies were produced against

antigen that did not contain ubiquitin or ubiquitin-conjugated proteins. With the

antibodies, the presence of aldolase B epitopes in a major 68 kD ubiquitinated protein

(Figs. 1-3 and 1-4, Ub68) and other ubiquitin conjugates was confirmed in subcellular

fractions from rat liver. Epitope-tagged aldolase B was expressed in E36 (parent) and

ts20 (ubiquitination mutant) cells previously used to establish ubiquitin-dependency for










heat stress-induced autophagic degradation of long-lived proteins. By examining

changes in the endogenous aldolase A and exogenous aldolase B associated with

pelletable subcellular fractions, evidence was found that these aldolase isoforms require

ubiquitination for autophagic degradation in lysosomes during heat stress. This

supported my hypothesis that stress-induced degradation of aldolase B requires

ubiquitination.

An attempt was made to use protein degradation measurements to confirm that

heat stress-induced degradation of aldolase B requires ubiquitination. Degradation of

aldolase B was found to utilize a temperature-dependent cytosolic proteolytic

mechanism. The cytosolic proteolysis of aldolase B at heat stress temperatures was

similar in magnitude to induced ubiquitin-mediated autophagic degradation. Since the

cytosolic mechanism turned out to be ubiquitin-independent, degradation measurements

could not confirm ubiquitin-mediated degradation of aldolase B via lysosomes.

However, the results demonstrate that mechanisms for degradation of aldolase B include

a novel cytosolic proteolysis.

My second aim was to test whether a receptor-mediated targeting signal was

required for stress-induced degradation of aldolase B. A sequence motif has been

defined for targeting cytosolic proteins to lysosomes for degradation during nutrient

deprivation, and aldolase B contains three sequences that match the motif (Fig. 1-2).

Depriving liver-derived cell lines of serum and amino acids causes starvation-induced

degradation of long-lived proteins including aldolase B. Vectors were constructed

expressing epitope-tagged aldolase B and used site-directed mutagenesis to disrupt the









32
putative targeting signals. Wildtype and mutant aldolase B proteins were expressed and

assayed for starvation-induced degradation. Starvation causes enhanced autophagic

degradation of aldolase B expressed in cultured hepatoma cells, and this enhanced

degradation specifically required a targeting signal that includes glutamine residue #111.

This supported my hypothesis that stress-induced degradation of aldolase B utilizes a

receptor-mediated targeting signal.














CHAPTER 2:
MATERIALS AND METHODS

Cell Lines and Culturing
General Maintenance

Except for temperature (see following subsections), all cell lines were maintained

similarly using standard sterile cell culturing techniques. Except where indicated, all

supplies were obtained from Fisher Scientific, Inc. The term "standard culture

conditions" refers to maintenance in DMEM (Sigma #D-5648), 2.2 g/1 NaHCO3, and

10% FBS (Biocell #6201-00) in a 5% CO2 atmosphere, and the standard medium for

stably transfected cells included 0.3 mg/ml active G418 (GIBCO BRL #11811-031).

Cultures were fed every 3-4 days and passage before complete confluency. For

passages, cell sheets were rinsed with DPBS (Sigma #D-5652) followed by lx trypsin-

EDTA (Sigma #T4174) in DPBS for 4-8 minutes at room temperature or 37C as

needed. Passages to amplify and maintain cultures for experiments were split 1:10 to

1:50 (area:area), and very fast growing lines that tolerated thin splits were done down to

1:80. Since trypsin/EDTA diluted 1:10 or more with 10% FBS did not affect cell sheets

during 30 min. at 37C, some thin splits (at least 1:30 into medium with 10%FBS) were

directly plated without pelleting to remove trypsin. For cultures using this short cut,

attachment times, spreading times, growth rates, and experimental results were

unaltered. Passages to replenish frozen stocks were always split heavily at 1:3 to 1:6

from freshly thawed stocks grown to near confluency. For new frozen stocks, cells
33








34
were suspended in medium supplemented with 10% DMSO (Sigma #D-2650), incubated

1-2 h at minus 20C, then at minus 80C overnight, and stored at minus 80C for up to

two months or transferred to liquid nitrogen for longer storage times.

Heat Stress-Inducible E36 Cells and Ubiquitination Mutant

Alan Schwartz kindly provided cell lines: E36 (parent), ts20 (mutant with

temperature sensitive ubiquitin-activating enzyme El ), and ts20E 1 c2 (mutant rescued by

wild-type human El) Chinese hamster lung cell lines. These cells are well characterized

for thermal control of ubiquitin-activating enzyme El activity and together have

demonstrated that El-mediated ubiquitination is required for heat stress-induced

degradation of long-lived proteins ((Handley-Gearhart et al., 1994); (Handley-Gearhart

et al., 1994); (Trausch et al., 1993); (Lenk et al., 1992); (Schwartz et al., 1992);

(Gropper et al., 1991); (Kulka et al., 1988)).

Starvation-Inducible Cell Lines

William A. Dunn, Jr. provided Fao (rat hepatoma) and HuH7 (human hepatoma)

cell lines. The Fao cell line originates from a rat hepatocellular carcinoma (Reuber,

1961), and this derivation is well documented (Deschatrette and Weiss, 1974). Fao cells

retain a dozen liver-specific characteristics examined by Mary C. Weiss, including some

endogenous expression of aldolase B (Deschatrette et al., 1979; Deschatrette and Weiss,

1974).

The HuH7 cell line was isolated from a well differentiated carcinoma of a

Japanese man and shown to secrete 16 different plasma proteins associated with liver










function (Nakabayashi et al., 1982). Seven expected carbohydrate-metabolizing

activities were present in HuH7 cells, but for two of these, liver-specific isoforms,

pyruvate kinase L and a low-Km hexokinase, were not detected (Nakabayashi et al.,

1982). BHK (baby hamster kidney), and NRK (normal rat kidney) cell lines were

examined briefly during transient transfections.

Plasmid Vector Construction and Mutagenesis

General Molecular Biological Methods

Basic methods were performed essentially as described in Current Protocols in

Molecular Biology (Ausubel, et al., 1994). Except where noted, all supplies came from

Fisher Scientific, Inc. Kits for DNA preparations were from Qiagen and Promega.

Restriction digestions, ligations, other DNA modifications, and PCR utilized supplies

from Promega and New England Biolabs, except as noted below.

PCR Primers and DNA Sequencing

At the University of Florida, the DNA Synthesis Core Laboratory provided all

oligonucleotide primers that William A. Dunn, Jr. or Peter P. Susan designed for PCR.

The University of Florida DNA Sequencing Core Laboratory sequenced parts of

plasmid vectors that were altered, or we did DNA sequencing with a SequenaseTm kit

(U.S. Biochemical Corporation).

Expression Vectors for Epitope-Tagged Aldolase B

Kiichi Ishikawa provided pRAB 1710 Amp+ (Tsutsumi et al., 1984), a plasmid

containing the cDNA of rat aldolase B (RAB) used as a PCR template in a reaction

containing two primers (Fig. 2-1). PCR solutions were prepared according to










manufacturer's specifications (Promega) and then run through 40 cycles (each cycle: 1

min. at 94C, 1 min. at 52C, and 2 min. at 72C), yielding DNA coding for rat aldolase

B tagged with the 9E10 myc epitope at the carboxyl terminus (RABM). After

restriction, the product was ligated into XhoI and XbaI sites of the vector pMAMneo-

blue (CLONTECH), yielding pRABM which failed to express RABM. Using EcoRV

and XbaI, I transferred the RABM code to pcDNA3 (Invitrogen), yielding

pcDNA3RABM (Fig. 2-2). William A. Dunn, Jr. provided a pcDNA3-based vector,

pHAHAB which expresses human aldolase B (Sakakibara et al., 1989) with an amino

terminal 12CA5 HA epitope, HAHAB (Lenk et al., Submitted 1998).






a. WID5, 5' primer: CTCCCTTGGCTCGAGCTGTC
XhoI


Xbal
b. WID9, 3' primer: TGCTCTAGACTActacaagtcttcttcagaaataagcttttgttcctcGTAGG-
TGTAGGGGCTGTGA



Figure 2-1: Primers for PCR Amplification of Insert Containing cDNA for RABM
Expression. a) 5' primer also called, WID5; b) 3' primer also called WID9. Orientations
are relative to 5' to 3' convention. Italics indicate reverse complementary code for
carboxyl terminal amino acids; lower case letters indicate reverse complementary
sequence for myc (9E 10) tag; single underline indicates reverse complementary sequence
for a stop codon; boldface indicates XhoI and XbaI restriction sites; other bases are the
same as vector sequences.










a. 5' DNA Sequence:


--TCTGCAGATATCAAGCTTA TCGA TA CCGTCGACCTCGAGCTGTCAATCAIT.--
EcoR V XhoI start
methionine
(aldolase B)



b. 3' DNA Sequence:

myc-epitope code
--ACCTACgaggaacaaaagcttatttctgaagaagacttgTAGTCTAGAGGGCCC--
C-terminal stop XbaI
tyrosine codon
(aldolase B)



C. Carboxyl terminal amino acids of RABM:

--TASYTYEEOKLISEEDL



FIGURE 2-2: DNA Sequence ofpcDNA3RABM. a) 5' insertion site and b) 3' insertion
site showing new sequence generated by vector construction. Boldface designates
cDNA sequence of rat aldolase B (upper case) and human myc epitope 9E10 (lower
case). Italics designate DNA sequence from the multicloning site ofpMAMneo-blueTM.
Standard typeface designates DNA sequence in the multicloning site ofpcDNA3TM
(Invitrogen). Underline designates indicated restriction sites. Double underline
designates indicated codons. Sequences not shown for rat aldolase B cDNA and
pcDNA3TM vector are available in Tsutsumi, et al. (Tsutsumi et al., 1984) and from
Invitrogen Technical Services, respectively, c) Amino acid sequence predicted for the
carboxyl terminus of the RABM protein expressed from this vector (residues not shown
would be identical to rat aldolase B), single letter amino acid code is underlined for
residues added to create a 9E10 epitope.










Site-Directed Mutagenesis of pcDNA3RABM

I used three different strategies for PCR mutagenesis: (1) overlap extension (Ho

et al., 1989), (2) Quick-ChangeTM Site-Directed Mutagenesis Kit (Stratagene #200518),

or (3) restriction-limited insertion as described below (Fig. 2-4; Table 2-1).

I used an overlap extension protocol adapted by Brian Cain from Ho and others

(Ho et al., 1989). In brief, a mutagenic primer pair (see Fig. 2-3, positions 2 & 3, 4 & 5,

or 6 & 7) was made complementary to each other and to base pairs on either side of a

targeted change (non-complementary) in aldolase B coding sequence (Table 2-1). For

example, Q58 mutagenesis started with pcDNA3RABM as template in two PCR

reactions using primers at positions 1 & 2 (Fig. 2-3) to make product for the 5' end of an

insert and at positions 3 & 8 (Fig. 2-3) to make product for the 3' end of an insert. PCR

was done for 35 cycles (1 min. at 94C, 1 min. at 55C, and 2 min. at 72C).

At one end of one product, sequence was derived from primer at position 2 and

therefore was reverse complementary to one end of the other product derived from

primer at position 3. These products were combined in Taq polymerase buffer

(Promega), melted at 94C, and cooled very slowly to allow promiscuous annealing and

yielding a small amount of 3'end-to-end annealed single stranded DNA from 5' and 3'

products for ends of a desired insert. Taq polymerase was added and the temperature

raised to 72C for run-off extension through ends complementary to positions 1 & 8

(Fig. 2-3). Primers for positions 1 & 8 were added and cycled through temperatures as

done previously which produced a smear of products due to promiscuous annealing.













Rat AldolaseB


1 3
4^I 1'3


5 7


I I -- -- -- --
B E 2 4 6 B b


FIGURE 2-3: Positions of Primers for Site-Directed Mutagenesis. Map shows the RABM coding region
ofpcDNA3RABM; positions drawn to scale. Numbered arrows identify 5'-> 3' primer sequences at
complementary sites. Shaded areas are cDNA sequences for the indicated polypeptides. Letters B, E,
and Xb indicate restriction sites for enzymes BsaI, EcoR V, and Xba I, respectively. E and Xb are sites
of insertion into pcDNA3 (Invitrogen) multicloning site.


TABLE 2-1: Details of Primers for Site-Directed Mutanenesis


Position (bp)
Primer Map From Target Primer Sequence, 5'to 3'
I.D. Posi- 1St Base of Code (sequence non-complementary
_____ tion* Start Codon Change to pcDNA3RABM is underlined)
WID17 1 -117 to +SmaI CTCACTATAGGGAGACCCGGGCTTGGT
-91______________
WID19 2 +25 to Q12(T/N) CTCCTTrCTTA(T/G)TCTCTGAGGT
+45______________
WID18 3 +25 to Q12(T/N) ACCTCAGAGA(A/C)TAAGAAGGAG
+45
WID21 4 +163 to Q58(T/N) CTCGGAA'/G)TCCTTCGGT-
__________+181___________
WID20 5 +163 to Q58(T/N) AACCGAAGGA(A/C)ITCCGAG
__________+181____________
WID44 4- +165 to +ScaI AGAGAAGTACTAAAGAGGAGCTCTCGG
non*** +195 Q58N AAATTCCTTCGG
WID43 5- +196 to +PmlI CGAGACACGTGGACAATTCTATC
_____non*** +218
WID23 6 +322 to Q11 1(T/N) ACCTCCA(GT)TGTCCAGCTT
~________+339_____________
WID22 7 +322 to Q111 (T/N) AAGCTGGACA(A/C)TGGAGGT
______+339 ______________
WID36 6 +313 to QUIT CACCTCCTGTGTCAAGCTTGATGCCCAC
+340 +HindlII___________
WID35 7 +313 to +HindIII GTGGGCATCAAGCTTGACACAGGAGGTG
_________+340 Q1I1TI
WID24 8 +617 to none AGCAGCCAAGACCTTCTCAG
________+636 _______________________________
*Fig. 2-3;**amino acid (single letter code) change by residue # (start M = 1); -**primer non-
complementary to paired primer with position shifted for restriction-limited insertion (Fig. 2-4).








40
Though extraneous products were common, the most abundant product was the desired

mutated DNA fragment representing full length mutated insert. This insert containing

altered DNA code was BsaI digested, gel purified, and ligated into the corresponding

site of fresh pcDNA3RABM from which the wild type fragment was removed.

Normally, restriction sites for primer positions 1 & 8 would be designed for two unique-

site enzymes producing different overhangs. However, BsaI cuts outside its recognition

sequence producing randomly unique overhangs that abrogate a need for separate

enzymes. BsaI cuts a third site near the ampicillin-resistance gene of pcDNA3RABM,

producing two fragments of vector besides the insert fragment. Overhangs for all three

sites were randomly different allowing three-fragment ligation with proper orientations.

The Quick-ChangeTM Site-Directed Mutagenesis Kit (Stratagene) was also used

and found to be more rapid. The manufacturer's protocols were followed, and primers

designed for primer extension and amplification at QI 11 (WID23 and WID22 at

positions 6 & 7) did not work with the Quick-ChangeTM kit. However, longer primers

(WID36 and WID35) were successful. The Quick-Change protocol involves in vitro

synthesis of the entire vector (6.5 kb), possibly introducing errors anywhere in mutated

pcDNARABM. To reduce sequencing, the BsaI fragment containing new mutations was

cassette into fresh vectors. All altered regions of vectors were sequenced at least twice

to confirm changes in amino acid coding were specific for targeted residues.

For Q58N mutation, a restriction-limited insertion was designed(Fig.2-4). This

method uses a mutagenic primer (WID44) to span the Q58 codon and code for unique










blunt-end restriction site, Scal (restriction-limited). Another primer (WID43) was

designed with another unique blunt site, PmlI, such that ScaI to Pml I blunt ends ligated

to make proper aldolase B code. Steps were followed as indicated in Figure 2-4 to

produce an expression vector for the mutated RABM, pcDNA3RABMQ58N.

Expressing Epitope-tagged Aldolase B in Cell Lines

Permanent Lines Expressing RABM

E36 and ts20 cells were transiently transfected with pcDNA3RABM, using Lipofectin

(GIBCO BRL) or DOTAP (Boehringer) by the manufacturers' protocol. When

transfected cultures approached confluency labeled cells occurred in groups of 2 to 8

presumably due to cell division. Transfected cultures were trypsinized at confluency and

diluted >1:15 by area into G418 Medium. Fresh G418 Medium was provided every 1-2

days as needed to remove cell debris and maintain strong selection. By 2-3 weeks post

plating, colonies of resistant cells were isolated, passage, and screened for RABM

expression immunofluorescencee microscopy and western blotting with anti-9E10

monoclonal antibody as described below).

Different lines permanently expressed RABM at varied levels (-10 fold range on

western blots). Using experimental protocols described below, no effect was seen with

doubling time, degradation of RABM, degradation of total protein, or viability. Of eight

positive clones that continue to express after culture amplification, seven (4 from E36

and 3 from ts20) maintained stable relative levels of RABM for twenty additional

continuous doublings or longer (by immunofluorescence and on western blots, data not

















target codon
for Q58
I


WID5 | UjUAUAAlN.Ai. -0
Xho Il I
X1 GGCTTCC TAAAGGCTCTCGAGGAGAAATCAT C
W ED44 / AGAGA
I Sca 5'


1. PCR with WID5 and WID44
2. Digest with Xho I and Sca I


TCGAGCTT A O'_ITTTAGT
CGA^^' AAAICA GTGGACA'
'AGCT CACCTGT

3. Ligate to make pcDNA3RABMQ58N


1. PCR with WID44 and WID9
2. Digest with Pml I and Xba I


/1 TACACrC"N%


pcDNA3RABM digested with Xho I and Xba I


Figure 2-4: Restriction-Limited Insertion for Constructing pcDNA3RABMQ58N.
Above, primers (small arrows and sequences for WID43 and WID44) are shown relative
to pcDNA3RABM template (bar). Sequences juxtaposed to template indicate
complementary regions. Relative positions of Q58 codon and restriction sites are
indicated. Below, DNA pieces for a three-fragment ligation. See text, Fig. 2-3, and
Table 2-1.


Pm] I
5 GAA/ W3D43
AG .~c/ .. ..... '


' pcDNA3RABM
or template
wMx









43
shown). To facilitate experimental quantification, the highest RABM-expressing E36

and ts20 cell lines fully designated E36RABM14.1 and ts20RABM10.2 or abbreviated

throughout this dissertation as E36AB and ts20AB, respectively, were used. In control

experiments, variation in the level of RABM expression did not affect results (data not

shown).

Similar procedures were repeated with Fao rat hepatoma cells transfected, except

another plasmid pHAHAB also was used to express human aldolase B tagged with the

12CA5 HA epitope at its amino terminus (HAHAB). Screening for HAHAB expression

was done with monoclonal antibody against 12CA5. When G418-resistant clones were

isolated at very most 20% of cells in a given clone had visible expression that was mostly

dim with a few bright cells, and this fraction was rapidly lost with culture splitting for

amplification. Subcloning and screening of a few hundred colonies produced one truly

stable line expressing HAHAB at levels comparable to ts20AB expression of RABM.

This line was designated FaoAB. FaoAB cells split at low density (1:20 or less) grew

much slower than parent Fao cells. In passages using about 1:10 or 1:15 splits, growth

rates were similar to parent Fao cells. An attempt to isolate HuH7 cell lines expressing

RABM was made, but failed to produce any clones having permanent expression.

Transient Transfection System

Fao, HuH7, NRK, and BHK (hepatic and renal cell lines derived from tissues that

express aldolase B) were transfected for transient RABM expression with a series of

lipids according to manufacturer's standard protocols (Invitrogen and Boehringer-









44
Mannheim). Transient transfection gave very broad cell-to-cell variation in expression by

9E10 immunofluorescence, but ratios of bright to dim cells were relatively reproducible

between transfections. Transfection efficiency was defined as fraction of labeled cells.

Immunofluorescence

Cells were grown on glass coverslips to desired confluency, rinsed briefly with

PBS and fixed with 4% paraformaldehyde in PBS for 20-30 minutes. Fixed cells were

washed three times for 10 minutes in 50 mM ammonium chloride/ 0.1% Tx-1 00/PBS.

Coverslips were placed on drops containing antibody diluted 1:100 in 5%NGS/ 0.1%

Tx-100/PBS for 1-2 hours at room temperature. Coverslips were washed four times for

5 minutes in 0.1% Tx-100/PBS. Coverslips were placed on drops containing an

appropriate secondary antibody (rhodamine or fluorescein conjugated) diluted 1:100 in

5%NGS/ 0.1% Tx-100/PBS for 1 hour at room temperature. Coverslips were washed

six times for 5 minutes in 0.1% Tx-100/PBS, then mounted on Fluoromount G (GIBCO

BRL).

Antibodies

Preparation of Ubiquitin-Free Aldolase B Antigen

Different E. coli strains were transformed with pXPB, a plasmid vector kindly

provided by Dean R. Tolan for bacterial expression of enzymatically active human

aldolase B (Beemrnink and Tolan, 1992). The aldolase B expressed in E. coli retains all

the enzymatic properties of the protein isolated from human liver (Sakakibara et al.,









45
1989). Increased expression of a 40 kD Coomassie signal in SDS-PAGE of whole cell

preparations was apparent in transformed cells.

E. coli JM83 cells were transformed with pXPB, which produced much more 40

kD protein than untransformed cells, about 0.3 mg per ml of 1.9 OD600nm culture. A 250

ml culture of LB broth + ampicillin (10.tg/ml) was grown to 1.9 OD6o0 nm culture and

pelleted in a Beckman GSA rotor at 3500 rpm for 10 min. Samples were maintained at

0-4C for the rest of the procedure. The cell pellet was resuspended in 20 ml 15%

sucrose/50 mM EDTA/50 mM Tris-HCl pH 8.5. To this, 5 ml 5 mg/ml lysozyme was

added, gently mixed by inverting, and incubated 15 min. Then 15 ml 0.1% Triton X-

100/50 mM Tris-HCl pH 8.5 was added, gently mixed, and incubated with periodic

inverting for 20 min. After centrifugation at 9000 rpm for 30 min. (GSA rotor), the

supernatant was decanted into Polyclear tubes and ultracentrifuged in aSW27 rotor at

23,000 rpm for 60 min. The resulting supernatant was the crude extract which was

further processed as previously described for isolation of aldolase B from liver extracts

(Penhoet and Rutter, 1975). Saturated (NH4)2SO4 was slowly added (0.5 ml/min.) to

45% final concentration with constant stirring. After centrifugation at 9000 rpm for 60

min., the supernatant was collected and the pellet discarded. (NH4)2SO4 was slowly

added to 60% concentration, and 6 N NH4OH added to pH 7.5. The mixture was

immediately transferred to centrifuge bottles and let stand for >2 hours. After

centrifugation at 9000 rpm for 60 min., the 60% (NH4)2SO4 pellet was dissolved in 1

mM EDTA/10 mM Tris-HCl pH 7.5 and dialyzed against the same buffer. The sample










was loaded onto a 25 ml phosphocellulose (fine mesh, 1.26 meq/g) column prepared

exactly according to Penhoet and Rutter. The column was washed with 5 mM EDTA/50

mM Tris-HCl pH 7.5 (50-60 ml) until OD280 m approached zero. Then 2.5 mM fructose

1,6- diphosphate in 1 mM EDTA/10 mM Tris-HCl pH 7.5 was used to specifically eluted

a sharp aldolase peak. Peak fractions with specific activities of 0.83 to 0.98 aldolase

U/mg (1 U/mg expected for aldolase B) were pooled, precipitated by 55% (NH4)2S04,

and stored as a suspension at 4C. Before immunization, the suspension was dialyzed

into 10 mM Tris-HCl pH 7.5. According to Coomassie labeled SDS-PAGE and

enzymatic properties (Rutter et al., 1966), the recombinant human aldolase B constituted

at least 95% of the final protein and was more than 99.99% pure of bacterial aldolase

activities, containing <0.005% bacterial isoform (EDTA-sensitive) activity.

Production of Antibodies Against Aldolase B

Rabbits were fed and housed by University of Florida Laboratory Animal

Services. Antibodies to native and denatured aldolase B were raised as previously

described (Reznick et al., 1985) except ubiquitin-free human aldolase B antigen was used

(as prepared above). For making antibody to native aldolase B, 50 gg antigen in 0.5 ml

10 mM Tris-HCl pH 7.5 was emulsified 1:2 with complete and incomplete Freund's

adjuvants for immunizations and boosts, respectively. For antibody to denature antigen,

the antigen solution was supplemented with 2%SDS/2%O3ME and boiled 10 min. prior to

mixing with adjuvant and immediately before administration to animals. Intradermal










injections were in the thoracic region on the backs of specific pathogen-free New

Zealand White rabbits. The first boost was two weeks after initial immunization.

Preparative Western blots of rat liver cytosol were routinely used to follow

specific immunoreactivities. One week after the first boost, the rabbit receiving native

antigen produced a highly reactive serum specific for 40 kD aldolase B which was

maintained without further boosts. Unless otherwise specified, these antibodies were

used to detect native aldolase B-specific polyclonal epitopes throughout this study.

One week after the first boost, the rabbit receiving denatured antigen produced

antibody that specifically labeled a 68 kD protein moderately, 60 kD and 78 kD proteins

lightly, and a high molecular weight smear. This pattern was remarkably similar to that

for anti-Ub68. Furthermore, relative recognition ofisoforms A and B were comparable

to that for Ub68. Thus, denaturation of aldolase B disrupted isoform-specific epitopes

and produced anti-denatured aldolase B that similarly recognized epitopes in both

aldolases A and B. Interestingly, the early bleeds had little or no reactivity to 40 kD

aldolase.

Every 4-7 weeks (after injection sites completely healed), the rabbit immunized

with denatured aldolase B was boosted. After the second boost, antibody

immunoreactivities were greatly increased. Though these sera recognized some non-

protein epitopes, they retained specificity for proteins labeled with sera from earlier

bleeds. After the third boost, sera recognized 40 kD aldolase B but never as well as

antibody to native antigen.










Other Antibodies

Anti-Ub68 was provided by William A. Dunn, Jr. and is a polyclonal rabbit

antibody raised against a major ubiquitin-protein conjugate purified from lysosomes

(Lenk et al., Submitted 1998). Monoclonal mouse antibodies against thel2CA5 HA and

9E 10 myc epitopes were obtained from the University of Florida Hybridoma Core

Laboratory. Alternatively, hybridoma cells expressing anti-9E10 (provided by the

Hybridoma Core) were injected into the peritoneum of BALB/c mice (provided by the

University of Florida Laboratory Animal Services Division), and periodically, ascites was

harvested until mice showed signs of discomfort or disease at which time they were

euthanized.

Viability Assays

All treatments were measured for viability except for incubations in HBSS

(Hank's Balanced Salts Solution) which were used to induce cell death. Heat stressed

cultures using HBSS had many cells rounding and sloughing off culture surfaces in 4 to 6

hours of treatment, and were only used in experiments to determine the effect of dying

cells on protein degradation measurements. When HBSS was replaced with MEM

(Sigma #M-0268 + 2.2 g/1 sodium bicarbonate), such rounding and sloughing was

delayed beyond 25 hours of treatment. To measure metabolic viability, cultures exposed

to each experimental treatment were recovered under normal maintenance conditions for

12 hours followed by addition of the same labeling medium (0.1 mCi 35S-methionine/ml)

used for protein degradation assays. Then protein synthesis was measured as TCA










precipitable counts incorporated in 20 min. Metabolic viability was defined as the %

protein synthesis relative to duplicate cultures treated with fresh medium under normal

maintenance conditions. Since experimental treatments (see Experimental Conditions)

lack serum and nutrients (MEM instead of DMEM) relative to maintenance conditions,

our assay probably underestimates metabolic viability. In MEM, more than 80% of

metabolic viability was retained for 28 hours and 15 hours in heat stressed E36 and ts20

cells, respectively. Unless otherwise stated, all data are reported for incubations and

drug treatments that retained 90% or greater metabolic viability. For low density

cultures (<10% confluent), the Cell Titer 96 AQ System was used according to

manufacturer's protocols (Promega) which indirectly measures electron transport

pathway activity.

Subcellular Fractionation

Subcellular fractionation of E36 and ts20 cells is summarized in Figure 2-6. To

produce a homogenate containing intact organelles, cells were grown to recent

confluency in 100 mm dishes, using 10 ml DMEM (Sigma #D-5648) + 10% FBS

medium. Culture rinsed with 10 ml DPBS (Sigma D-5652) was treated with 1.0 ml 1 X

Trypsin-EDTA (Sigma #T-9395 diluted in DPBS),and incubated at room temperature

until cells easily and completely knocked loose from the plate (10 minute maximum).

After adding, 5 to 10 ml medium containing 10% FBS, cells were transferred to 15 ml

conical centrifuge tubes (polypropylene), centrifuged at 1,500 X g for 5 minutes, and

supernatant discarded. Pellet was suspended in 1.0 ml ice-cold cavitation buffer (SHE):







Figure 2-6: Subcellular Fractionation Scheme for E36 and ts20 Cells. 50


Cell Culture


Scrape cell sheet

Centrifuge 1000 x g


-> Supernatant = Sc


Pellet


Homogenize
4


--> Homogenate = Ho


Centrifuge 1000 x g
$
Supernatant

Centrifuge 1000 x g
S
Supernatant = L
4-


Centrifuge 100,000 x g
4,.
Pellet
4,.
Resuspend

Centrifuge 100,000 x g


-> Pellet
4-
Pool = LP
T
-* Pellet


-> Supernatant = HS


-> Pellet = HP


Supernatant (wash, dilute HS) --> <1% of culture content








51
250 mM sucrose, 10 mM HEPES (Research Organics #6003H-3), 1 mM EDTA, pH 7.4,

loaded into a N2-cavitation bomb chamber using 2-3 ml total SHE volume, pressurized

to 65 psi for 10 minutes, and collected sample from bomb directly into a pre-chilled

Dounce homogenizer. Procedural details were as described by the cavitation bomb

manufacturer's specifications (Kontes). The sample was homogenized with 5 strokes of

a pestle, minimizing froth by limiting passage of bubbles to the sample side of pestle.

This was saved as the homogenate (Ho).

Alternatively, the trypsinization step was replaced by scraping the cell sheet

directly into SHE which resulted in a large fraction of cytosol but not organelles to leak

out. This facilitated the separation of aldolase associated with organelles from soluble

aldolase in the cytosol. Scraped cells were pelleted as done above to remove trypsin

solution, but in this case the supernatant was saved (to assess cytosolic leakage) as the

scrape fraction (Sc). The rest of cavitation and homogenization was performed as

above.

Homogenate (Ho) was fractionated essentially as described previously

(Rickwood, 1992; Coligan et al., 1995), using Sigma reagents for assays and Sorvall or

Beckmann centrifuges and accessories. Homogenate was centrifuged at 1,500 X g for

20 minutes yielding a low speed pellet (LP) and supernatant. The centrifugation was

repeated with the supernatant to make sure nuclei, large debris, and unbroken cells were

efficiently removed, the resulting pellet was pooled in LP, and the resulting low speed

supernatant (LS) was centrifuged at 100,000 X g for 90 minutes, yielding a high speed









supernatant (HS) and pellet. The pellet was resuspended in well over 400 volumes of

fresh SHE and centrifuged at 100,000 X g for 60 minutes. The supernatant had a

content similar to HS but was much more dilute (data not shown), so it was not pooled

with HS. The pellet was saved as the high speed pellet (HP).

Fractionation conditions were developed to separate abundant cytosolic aldolase

from that associated with organelles. An initial scrape into fractionation buffer caused a

three fold greater leakage of aldolase than acid phosphatase, so this step was retained in

the procedure. Conditions were chosen to maximize recovery oflysosomal organelles

(acid phosphatase and 3-hexosaminidase) from LP to HP and minimize release of

lysosomal markers into Sc and HS. Recovery was reasonable (85-99% accounted).

Enzyme Assays

Aldolase

Aldolase assays were performed as previously described (Penhoet and Rutter,

1975). "Aldolase reaction mix" includes 50 p1 6.3 mg/ml a-glycerophosphate

dehydrogenase-triose phosphate isomerase mixture + 4 mg NADH (99% pure, Sigma) +

20 ml 0.1 M glycylglycine pH 7.5. Add 5-50 pl of sample to 1 ml of aldolase reaction

mix, and measure background AOD (340 nm)/min. (BG); add 50 pl 50 mM fructose-1,6-

diphosphate (FDP) or 100pl 100 mM fructose-I-phosphate (F-l-P), and measure assay

AOD (340 nm)/min. (ASSAY). Aldolase activity in units, U = (ASSAY-BG)/12.44 for

FDP or = (ASSAY-BG)/6.22 for F-i-P.










Acid Phosphatase

Acid Phosphatase activity was the OD (405 runm) in 1000x g supernatant after 60

minute incubation at 37C for 50 ul sample in 200 pl 8 mM p-nitrophenolphosphate/

2mM MgC12/ 90 mM Na-acetate pH 5.0 stopped by 600 ul 0.25 M NaOH (Rickwood,

1992).

Protein Analysis

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel

staining and drying, western blotting, and autoradiography followed standard protocols

similar to those described and referenced elsewhere (Ausubel et al., 1994; Coligan et al.,

1995). Gels were made with 10% polyacrylamide (1:35 bis-acrylamide).

Immunochemiluminescent detection was done with Amersham ECL Reagents and

protocols, except blots were rinsed in Kodak 1XCDS buffer just before exposure to ECL

chemicals. Protein concentrations were determined with Bio-Rad protein assay (IgG

standard) reagent or spectrophotometric absorbence (205 nm or 280 nm).

For direct immunoprecipitations, monoclonal antibody was purified and

conjugated to sepharose 4b by a CNBr protocol similar to that described in Coligan et

al., 1995. Cells were incubated on a shaker at 0-4C with ice cold minimum lysis buffer

(MLB: 1%NP40/ 1 mM EDTA/50mM Tris-HCl pH 7.4), standard lysis buffer (SLB:

150 mM NaCl MLB), or modified radioimmunoprecipitation assay buffer (mRIPA:

0.1% SDS/ 0.25% desoxycholate/ SLB) supplemented with a cocktail ofprotease

inhibitors (leupeptin, TLCK, pepstatin, aprotinin, and PMSF obtained from Sigma or










Boehringer/Mannheim) at concentrations according to Harlowe. The mRIPA gave the

best results with mAb's conjugated to sepharose, and SLB gave the best results with

polyclonal sera precipitated with commercial protein A-agarose (Boehringer-Mannheim).

Lysates were precleared for 15-20 minutes on maximum in a microfulge then transferred

to tubes containing 4-10 ll packed bed volume of mAb-sepharose or 1-5 p.l antiserum

then rotated in the cold. After one hour, tubes containing antiserum only received 10 gl

packed bed of protein A-agarose. Rotating incubation was continued for varying times,

usually overnight, and such times were held constant within a given experiment. Rapid

washes by quick microcentrifugation and aspiration of supernatant were done three

times with the same lysis buffer and one time with TBS, then immediately processed for

other procedures. For SDS-PAGE, a 2-fold concentration of sample buffer was directly

applied to the pellet and heated at 95-100C for 5-10 minutes. For precipitable aldolase

activity, pellets were further washed with TE pH 7.5 then resuspended in aldolase

reaction buffer.

Densitometry autoradiographss, chemiluminographs, & Coomassie gels) were

quantified using a desktop scanner and Sigmagel software. Automatic brightness and

contrast settings determined initial settings that were then kept constant. Spot settings

were chosen to encompass regions of interest, reduce background, and optimize signal.

Standard curves were routinely performed to characterize linear response ranges for

relative protein levels which also defined backgrounds and allowed quantification of

relative signals. Where protein bands were specific for transfected cell lines,










untransfected cells were run in parallel through procedures and quantified to establish

backgrounds.

Stress-Induction of Protein Degradation

Culture Preparation

Cells were plated, grown, and maintained at confluency for 2-3 days. About 30-

40 hours before an experiment, cultures were fed with standard maintenance medium or

for protein degradation assays, 14C-Valine or "3S-Methionine was made up in comparable

medium with the corresponding unlabeled amino acid omitted. At the beginning of an

experiment, cells were switched to the media and temperatures indicated in Table 2-2.


Table 2-2: Comparison of Systems for Stress-Induced Degradation of Proteins
Heat Stress Induction Starvation Induction
MduControl Stress Control Stress
Medium MEM MEM DMEM+FBS KH*
Temperature 30.5C 39.5C 37C 37oC
*KH, Krebs-Heinseleit medium (Lefer et al., 1982)

Heat Stress

Cultures were prepared as described above and replicates were incubated under

control temperature (CT) and heat stressed (HS) conditions (Table 2-3) which are

permissive and non-permissive, respectively, for the ubiquitin-activating enzyme El of

ts20 cells (Handley-Gearhart et al., 1994; Kulka et al., 1988; Lenk et al., 1992).

Accordingly, CT conditions included 4 mM bicarbonate-buffered MEM under 5% CO2 at

30.5C, and HS conditions included 20 mM HEPES-buffered MEM under air at 41.5C

for 1 hour followed by 39.5C. These incubations followed established protocols for the









E36/ts20 cell system, except MEM replaced HBSS to improve viability. In some

experiments, media were supplemented with 5-10 mM 3-methyladenine (3MA) or 40-

160 .M chloroquine (CHQ) as indicated in Results. Logistics and consistency with

published protocols required differences in buffering and atmosphere between CT and

HS conditions. As a control for such


Table 2-3: Heat Stress (HS) and Control Temperature (CT) Treatments
to Determine Ubiquitin-Activating El Mediated Processes
Experimental
Condition MEM buffer* Temperature Incubator

CT 2.2 g/ 1 NaHCO3 30.5C standard
(Permissive) pH 7.4 5% CO2


HS 20 mM HEPES 1 hour @ 41.5C submerged
(Non-permissive) pH 7.4 then 39.5C in water bath

Repeat treatments with two cell lines: E36 (parent) and ts20 (mutant)
*For protein degradation experiments, medium was supplemented with unlabeled amino
acid corresponding to that used for labeling (5 mM methionine or 10 mM valine).
differences, CT as summarized in Table 2-3 was compared with CT in HEPES-buffered

MEM under air, yielding no differences in cell morphology, viability, or protein

degradation measurements (data not shown). The other control (i.e. comparing HS in

conditions above with HS in bicarbonate-buffered MEM under 5% C02) was not tested,

because bicarbonate buffering varies with temperature.

Starvation (Nutrient Stress)

Cultures were prepared as described above and replicates were refed with fresh

standard maintenance medium (DMEM + 10 % FBS) or Krebs-Heinseleit (KH) medium









57
and referred to as "Fed" or "Starved," respectively. KH components are given in Lefer,

et al., 1982. For protein degradation experiments, media were supplemented with

unlabeled amino acid corresponding to that used for labeling (5 mM methionine or 10

mM valine). Inhibitor treatments utilized the same levels as for heat stress above.

Protein Degradation

Permanent cell lines or transiently transfected cultures were treated according to

instructions in the section Stress-Induction of Protein Degradation to produce cells with

metabolically labeled proteins containing "S-methionine or 14C-valine. Cell sheets were

routinely rinsed with DPBS (Sigma) immediately followed by application of media

containing unlabeled excess amino acid (5 mM methionine or 10 mM valine) to cultures

described above. This initiated chase of radiolabel incorporated into proteins.

At various times, aliquots of media were collected and TCA precipitated to

measure release of soluble counts measured with a scintillation counter. At the end of

the chase, whole cultures were TCA precipitated to determine total counts. Fraction of

TCA soluble counts released at various times were subtracted from 1 to calculate TCA

precipitable counts, representing the remaining total radiolabeled protein at those times.

Alternatively, cultures were harvested at each time point, processed for

immunoprecipitation, SDS-PAGE, and autoradiography, and the radioactive signals in

specific protein bands (absent in untransfected cells) were quantified by densitometry,

representing the remaining radiolabeled protein (aldolase) at those times.








58
To compare degradative rates for proteins expressed at different concentrations,

relative rates are normalized to the initial amount of the protein, giving a fractional

change per time with units %/hour. This fractional rate is constant for unchanging

degradative mechanisms regardless of a substrate protein's concentration. For a given

protein, this fractional rate defines the degradative rate constant, kd. For total proteins,

the fractional rate represents weighted average kd contributed by the varied amounts of

different proteins. Throughout this dissertation, all degradative rates and other rates of

protein decrease are estimated with the following calculation. Fractions of radiolabeled

protein remaining were transformed by the natural log (In) and regression analysis

performed using the following function:


ln(100*St/So) = -kd t,


where St = signal at time t, So = initial signal, kd = first order degradative rate constant,

and t = time. Degradation rates were taken as the negative slope of the regression (kd)

and the standard error of the slope was calculated as the standard error ofy at x divided

by the square root of the deviations squared ofx. Degradative turnover is also described

by half-life, t%, the time needed to replace 50% of existing molecules with new ones. In

general, the degradative rate constant and half-life are simply converted by kd = ln(2)/ ty

= 0.693/ t. Note that some investigators do not follow the empirically confirmed first

order relationship of kd and ty. This results in kd and t% reported in the literature that can

vary by as much as three fold from similar data presented using conventional

calculations.















CHAPTER 3:
UBIQUITINATION MEDIATES LYSOSOMAL PROTEOLYSIS OF ALDOLASE B


Introduction

Aldolase B undergoes degradation during stress via autophagy. In Chapter 1,

evidence was described for ubiquitin protein conjugates which underwent starvation-

induced enrichment in autophagic vacuoles and lysosomes, and preliminary work

suggested that putative ubiquitinated aldolase B was amongst these conjugates (Figs. 1-3

and 1-4). This resulted in the hypothesis that stress-induced autophagic degradation of

aldolase B requires ubiquitination. In the first part of this chapter, the presence of

aldolase B in ubiquitin conjugates is confirmed, and a role for ubiquitination in the heat

stress-induced autophagic degradation of aldolase B is demonstrated.

Two mechanisms were found to simultaneously mediate enhanced degradation of

aldolase B during heat stress: autophagy and cytosolic proteolysis. To separately

examine effects on autophagy, heat stress-induced changes in endogenous aldolase A and

exogenous epitope-tagged aldolase B associated with pelletable organelles were assayed.

In addition, autophagic degradation of long-lived proteins was demonstrated to require

ubiquitination. The results described below support a role for ubiquitination in the

function of a subset of lysosomal proteases.










In Vivo Multiubiquitination of Aldolase B

Previously, antibodies were raised to a major ubiquitin-conjugated protein, Ub68,

that associated with autophagic vacuoles and lysosomes during stress-induced

autophagy. On western blots of subcellular fractions isolated from rat liver, anti-Ub68

produced a pattern suggestive of a 40 kD protein successively conjugated with serially

increasing numbers ofubiquitin. Such a pattern is referred to as a ubiquitin ladder for

the modified core protein. Peptide sequence analysis identified the 40 kD protein as

aldolase B, suggesting that anti-Ub68 reactive proteins might represent a ubiquitin ladder

for aldolase B.

To confirm the existence of ubiquitinated forms of aldolase B, antibodies (Fig. 3-

2) were raised against aldolase B (Fig. 3-1) and used to assay western blots of rat liver

fractions from the previous studies (Fig. 3-3). In short, anti-aldolase B recognized the

same proteins as anti-Ub68, confirming a ubiquitin ladder for aldolase B and

demonstrating that aldolase B is multiubiquitinated in vivo. The data are consistent with

ubiquitin-mediated autophagic degradation of aldolase B.

First, antibodies against aldolase B had to be produced. However, aldolase B

isolated from animal cells would need to be purified away from contaminating ubiquitin

and ubiquitin conjugates. Since bacteria lack ubiquitin, aldolase B was expressed in E.

coli and purified to produce ubiquitin-free antigen for immunization (Fig. 3-1). E. coli

strains transformed with an expression vector for human aldolase B (Fig. 3-la, lanes x)

produced more 40 kD protein (expected size of aldolase B) relative to untransformed

cells (Fig. 3-la,



















Figure 3-1: Isolation ofUbiquitin-Free Aldolase B Expressed in E. coli. a) Three E. coli
strains DH5c, JM109, and JM83 were transformed for aldolase B expression as
described in the text, pelleted, suspended in sample buffer, boiled, and run on SDS-
PAGE with 60 la culture (OD600 n = 1.5) equivalents per lane, u and x designate
untransformed and transformed cells, respectively. The 40 kD band specific for
transformed cells is indicated; b) E. coli JM83 cells expressing aldolase B were
fractionated as described in the text: 1 and 2, whole cell preparations (as in part a)
untransfected and transfected, respectively; 3, crude extract; 4, 45% (NH4)2S04
supernatant; 5, 45-60% (NH4)2SO4 cut; 6, aldolase activity peak from phosphocellulose
column; 7, dialyzed antigen ready for immunization; 8, rat liver cytosol used to screen
antibodies; 9, detection of lane 8 with antibody raised against protein in lane 7. Lanes 3
to 6 were loaded with 12 jig, lane 7 with 4 ug, lane 8 with 25 ug, and lane 9 with 10 uLg
of protein. Dark bands on light background indicate Coomassie R-250 label in gels, and
light bands on dark indicate western blotted proteins detected with anti-native aldolase B
by ECL (Amersham); c) Elution profile for phosophocellulose chromatography, Relative
Amounts: Protein, OD(280 nm); Aldolase Activity, mU/10 (loaded then started washes
when collecting fraction 9); FDP 4,, elution started with fructose 1,6-diphosphate.









DH5a JM109 JM83
a. u x u x u x


b. 1


2 3 4 5 6 789


H :. :-


40 00- I
O ---s f


5
4.5
4
3.5
3
2.5 -
2
1.5 -
1-
0.5
0


F jP )
"-L : I


0 5 10 15 20 25 30 35 40 45


Fraction Number


t -- Protein v A
--Aldolase Activity |


I


I























Figure 3-2: Antibodies Against Aldolase B. a) A preparative western blot of rat liver
cytosol run on SDS-PAGE (see Coomassie in Fig. 3-6b, lane 8) was prepared and strips
containing approximately 3 tg total protein were probed with sera from rabbits
immunized with native and denatured aldolase B as indicated, each lane contains a strip,
numbers above each lane correspond to bleed numbers, and numbers with arrowheads
indicate molecular weight, b) A Bio-Rad slot-blotting apparatus was used to load 10
ng/slot of antigen indicated by vertical labels (Aldolase A from Sigma), each row was
probed with serum raised against antigen indicated by horizontal labels with increasing
serum dilutions shown at the bottom of the figure, corresponding preimmune sera were
used in rows immediately above and below anti-aldolase B and anti-Ub68, respectively.








Native
0 1 13
a.

68 l

400 **


Denatured
0 1 2 3 4 5 6 7 8 9 10 11 12 13


111 .fl.j


Serum
Preimmune
Aldolase B
UB68
Preimmune
Preimmune
Aldolase B
Ub68
Preimmune


468

440


-


1
200


1 1 1
800 3200 12800


b.

1|
























Figure 3-3: Aldolase B Ubiquitinated In Vivo is Enriched in Lysosomes. a) Preparative
SDS-PAGE of cytosol from starved rat liver was western blotted onto nitrocellulose
then cut into strips with ~3 tg protein/strip, and stained with early antisera against
aldolase B (Fig.3-2a, bleed 4) or Ub68 (bleed 5); b) Aldolase B antisera from bleeds
after booster injections (Fig.3-6a, bleed 10) were reacted with Cy (cytosol strips as
prepared above) or ML (similar strips using a lysosome-enriched fraction instead). N,
antiserum to active native enzyme; No, same as N withlO-fold longer ECL exposure; D,
antiserum to chemically denatured enzyme; I, antiserum to antigen extracted from
polyacrylamide gel slices; and P, preimmune serum. Arrowheads, molecular weights in
kD. Dots on rightmost edge indicate bands at molecular weights higher than expected
for 40 kD aldolase B subunit. Susan E. Lenk provided subcellular fractions defined as
follows: a 1,000 x g supernatant of rat liver homogenate was centrifuged at 6,000 x g;
the resulting pellet was enriched in lysosomes and contained mitochondria (ML); the
6,000 x g supernatant was centrifuged at 100,000 x g yielding a supernatant fraction
referred to as cytosol (Cy).











Cytosol
Aldolase B Ub68
a. N D P I P


681
o


Aldolase B
b N N No No D D
b. CyMLCyML Cy ML



68"0 Zk

'; 0 0't0* *


. .










lanes u). Aldolase B was purified from the most productive strain, JM83, by cellulose

phosphate chromatography (Fig. 3-1c) of a 45-60% ammonium sulfate cut (Fig. 3-lb,

lane 5) from crude lysate (Fig. 3-lb, lane 3). Cellulose phosphate chromatography

separates aldolase B by substrate affinity at the enzyme's active site, allowing

enzymatically active aldolase B to be specifically eluted with fructose 1,6-diphosphate,

FDP (Fig. 3-1c, peak at fraction #30). Aldolase B in peak fractions was at least 95%

pure based on Coomassie stained SDS-PAGE gels (Figure 3-lb, lane 6) and specific

activities ranging 0.95-0.98 U/mg (pure aldolase B = 1.0 U/mg). EDTA resistance of

purified aldolase B activity indicated that contamination by class II bacterial aldolase was

less than 0.005% (data not shown).

Aldolase B antigen described above was used to raise antibodies against native

and chemically denatured aldolase B as detailed in Materials and Methods (Chapter 2).

Preparative Western blots of rat liver cytosol were routinely used to follow specific

immunoreactivities (Fig. 3-2a). Native antigen produced a highly reactive serum specific

for 40 kD aldolase B. Anti-native aldolase B demonstrated minimal cross-reactivity

with aldolase A (Fig. 3-2b). However, this antibody aldolase B effectively recognized

aldolase B from different animal species(Fig. 3-2, a. 40 kD rat aldolase B, b. purified

human aldolase B).

A rabbit immunized with denatured aldolase B produced antibodies (Fig. 3-2a

"Denatured" bleeds 2 through 5) that specifically labeled a pattern indistinguishable from

that for anti-Ub68 on western blots of subcellular fractions from rat liver (compare Fig.








68
3-3, lanes D with Figs 1-3 and 1-4, anti-Ub68). This demonstrated aldolase B epitopes

in previously identified ubiquitin-protein conjugates and confirmed that aldolase B is

ubiquitinated in vivo. The antibody to native aldolase B was specific for the 40 kD

unmodified monomer. However, with ten-fold greater exposure times, even the anti-

native aldolase B detected a ubiquitin ladder (Fig. 3-3b, No). Preimmune sera failed to

label any proteins. .Antibodies raised against native aldolase B are known to be highly

specific (Haimoto et al., 1989). Given this, labeling with anti-native aldolase B provides

even stronger evidence that aldolase B is multiubiquitinated in vivo, and confirms that

Ub68 is probably a stable conjugate of the form: (aldolase B)i(Ub)4.

Multiubiquitinated Aldolase B is Denatured and Enriched in Lysosomes

Aldolase A and B can spontaneously refold to active enzyme after reversible

denaturation treatments (Horecker, et al., 1972; Beemrnink and Tolan, 1996). Anti-

denatured aldolase B preferentially recognized ubiquitinated (> 40 kD) forms, whereas

anti-native aldolase B preferentially recognized the unmodified (= 40 kD) form (Figs. 3-

2a and 3-3). These results indicated that ubiquitination inhibits spontaneous refolding of

aldolase B into native conformations. Reznick and Gershon also raised antibodies

against native and denatured aldolase B (Reznick et al., 1985). Their anti-denatured

aldolase B failed to immunoprecipitate catalytically active enzyme, but it effectively

precipitated smaller peptides resulting from proteolysis. They also found that native

aldolase B antibody failed to precipitate proteolytic fragments, but efficiently pelleted

aldolase B activity (Reznick et al., 1985). The results reported in this study confirm the









hypothesis that native epitopes of aldolase B require three-dimensional conformations

that mask denatured epitopes.

The 40 kD unmodified aldolase B predominantly occurred in cytosol (Cy)

fractions with <10% in lysosome-enriched (ML) fractions (Fig. 3-3b). Consistent with

previous results (Fig. 1-3), ubiquitin-aldolase B conjugates (bands >40 kD) were

enriched in ML fractions with less occurring in Cy fractions (Fig. 3-3b). Taken together,

the data suggest that ubiquitination can provide a mechanism for maintaining aldolase B

in a denatured conformation. This could contribute to enhanced degradation of aldolase

B by making degradative signals more accessible or by making the protein more

vulnerable to proteases.

Heat Stress-Induced Delivery of Aldolase A to Lysosomes Requires Ubiquitination

Above, ubiquitinated aldolase B was confirmed to contribute to ubiquitin

conjugates that are enriched in autophagic vacuoles and lysosomes during nutrient stress

(Figs. 1-3, 1-4, and 3-3). The results suggested a role for ubiquitination in autophagic

degradation of aldolase B. During heat stress, ubiquitin-dependent autophagic

degradation of long-lived proteins has been demonstrated in E36 Chinese hamster lung

cells (Gropper, et al., 1991; Handley-Gearhart, et al., 1994). Our results suggested that

aldolase B was a possible substrate for this mechanism. However, E36 cells express

endogenous aldolase A but not aldolase B (described later). Both aldolase A and B are

established substrates for autophagy (reviewed in Chapter 1), so aldolase A was

examined as a substrate for ubiquitin-mediated delivery to lysosomes during heat stress.










In addition to autophagy, temperature-dependent cytosolic proteolysis

contributes to increased protein degradation during heat stress (next chapter; Hough and

Rechsteiner, 1984). During autophagy, cytosolic proteins, like aldolase A, are

sequestered into organelles (autophagic vacuoles and lysosomes) that can be pelleted by

differential centrifugation. To measure effects specific for the autophagic pathway,

aldolase A activity associated with pelletable organelles was assayed.

To examine a role for ubiquitination in the stress-induced degradation of aldolase

A in lysosomes, a system developed by Schwartz and Ciechanover was utilized for

measuring ubiquitin-dependent degradation of long-lived proteins (Gropper et al., 1991).

During heat stress, E36 cells undergo enhanced autophagic degradation of long-lived

proteins. However, ts20 cells derived from E36 cells harbor a temperature-sensitive

mutation in ubiquitination. Heat stress is non-permissive for the mutation, so

ubiquitination and enhanced autophagic degradation is inhibited in ts20 cells. The

degradative phenotypes were confirmed and are presented at the end of this chapter.

FDP aldolase activity was used to follow endogenous aldolase A, and acid

phosphatase activity was used to follow organelles in subcellular fractions collected by

differential centrifugation as described in Materials and Methods (Fig. 3-4).

Fractionation was optimized to maximize organelles released from cells, indicated by loss

of acid phosphatase from low-speed centrifugation pellets (LP), and to maximize

organellar integrity, indicated by fraction of acid phosphatase retained in high-speed

centrifugation pellets (HP). Acid phosphatase occurs as both an integral membrane











35%
S30%

S25%
020%
S15%
0 10%
S5%
I-
0%


E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatment


70%


0
S60%
S50%
0 40%
I--
" 30%
0
C
. 20%
S10%
U.%
0%-


b. Acid Phosphatase Activity


Sum T



-LBP T1 TT[JT


E36
CT


E36 E36 ts20
HS HS+CHQ CT
Cell Line and Treatment


ts20
HS


Figure 3-4: Ubiquitin-Dependent Association of Endogenous Aldolase A with
Organelles. Aldolase (a.) and acid phosphatase (b.) reported as % total culture activity
(mean SD, n = 3 cultures) for subcellular fractions collected from E36 and ts20 cells
treated for 8.5 h as indicated; CT, control temperature; HS, heat stress; +CHQ, 80 WM
chloroquine; subcellular fractions were collected (Materials and Methods) and are
labeled only on the first set of three bars (E36, CT): LP, low-speed pellet (1000x g); HP,
high-speed pellet (100,000x g); Sum, total pelleted fractions (LP + HP); different from
CT, Student's t-test: *, p <0.06; **, p <0.03; ***, p <0.009; ****, p <0.0008.










protein and as a soluble matrix protein inside lysosomes. For organelles isolated here,

acid phosphatase activity (about 60% of HP) was released by freeze-thaw (data not

shown), indicating that much of it was soluble in E36 cell lysosomes and served as an

adequate indicator of organellar integrity.

E36 cells were incubated at control (CT) and heat stress (HS) temperatures and

subcellular fractions pelleted during differential centrifugation were characterized for

aldolase and acid phosphatase enzymatic activities (Figs. 3-4 and 3-5). Relative to CT,

HS treatment significantly increased aldolase activity distributed in pelletable fractions

isolated from E36 cells (Fig. 3-4a). During 8.5 hour incubations that were used,

partitioning of aldolase to pelletable compartments had to be faster than loss. This is

consistent with accumulation of nascent autophagic vacuoles peaking by 6 hours after

autophagic induction (Lawrence and Brown, 1992). Chloroquine (+CHQ) caused a

more significant increase in pelletable aldolase activity. The effect of chloroquine

suggests that lysosomal degradation contributes to aldolase A flux out of pelletable

organelles consistent with an autophagic mechanism. In support of this, accumulation

caused by heat stress and chloroquine (Fig. 3-5, HS+CHQ) corresponds to a 1.50.3%/h

(mean SD, n = 3) increase in the sequestration rate for aldolase A which was similar in

magnitude to induced autophagic degradation for total long-lived proteins (data

presented in a later section). The results support endogenous aldolase A of E36 cells

undergoing autophagic delivery to lysosomes during heat stress.











Aldolase Accumulation
3.0
S,*LP cHP
(0 _^
N 62.5
t..II
0.
S"'2.0 1**

u 1.5
'o


1.0

50.



0.0 1 ; I ;
E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatnment
Figure 3-5: Ubiquitination Mediates Lysosomal Accumulation of Aldolase A During
Heat Stress. Using enzyme activities collected for Figure 3-4, aldolase was divided by
acid phosphatase to indicate relative aldolase associated with organelles (mean + SEM, n
= 3); values were normalized to the 100,000 x g pellet (HP) of control temperature (CT)
to reflect aldolase accumulation relative to unstressed conditions. Labels are as in Figure
3-4. Student's t-test: **, p <0.03; ***, p <0.009.



Heat stress (HS) by itself failed to affect the distribution acid phosphatase activity

in subcellular fractions (Fig. 3-4b). This indicated that increase of aldolase A in pellets

was not due to redistribution of lysosomal organelles and supported the idea that the

aldolase A was undergoing enhanced accumulation. Chloroquine treatment (+CHQ) had

no effect on total pelletable acid phosphatase (Sum) but caused a redistribution of acid

phosphatase from high-speed pellets (HP) to low-speed pellets (LP). It is known that










chloroquine treatment causes lysosomes to swell (Glaumann, et al., 1986). As a weak

base, chloroquine accumulates in organelles proportional to their acidity, and mature

lysosomes are the most acidic organelles. The results here indicate that some lysosomes

became large enough to pellet at lower centrifugation speeds.

A basic result of subcellular fractionation is that different pelleted fractions have

different contents of organelles (Rickwood, 1992). To demonstrate that aldolase A

accumulates in a subpopulation of organelles (presumably lysosomes), aldolase activity

was normalized to acid phosphatase activity and calculated the accumulation of aldolase

A in HP and LP relative to lysosomal content (Fig. 3-5). Significant accumulation of

aldolase activity only occurred in HP fractions during heat stress. In the presence of

chloroquine (+CHQ), there was a greater than two-fold accumulation of aldolase activity

in HP fractions but not LP fractions. The results suggest that aldolase A containing

organelles were preferentially isolated in HP even during CHQ treatment, and are

consistent with heat stress causing accumulation of aldolase A in a subpopulation of

lysosomes.

A previous study has shown that the fractional volume of autophagic vacuoles

and lysosomes does not significantly increase in heat stressed E36 cells (Lenk, et al.,

1992). Together, the data indicate that heat stress increases the flux of aldolase A into

autophagic vacuoles during heat stress. Unlike wildtype E36 cells, heat stress-induced

accumulation of aldolase activity with pelletable organelles failed to occur for mutant

ts20 cells (Figs. 3-4 and 3-5). Since heat stress inhibits ubiquitination in ts20 cells, this









75
suggested that aldolase A accumulation in organelles requires ubiquitination. The data

support a role for ubiquitination in heat-stress induced sequestration of aldolase A.

Using electron microscopic morphometry, a previous study shows that in heat stressed

ts20 cells conversion of autophagic lysosomes into residual bodies is specifically

inhibited, resulting in a 6-fold accumulation of lysosomal volume (Lenk, et al., 1992).

The subcellular fractionation results here indicate that earlier events in autophagic

degradation (aldolase A sequestration) might also involve ubiquitination. In conclusion,

the endogenous aldolase A of E36 cells appears to require ubiquitination for heat-stress

induced delivery to lysosomes.

Heat Stress-Induced Lysosomal Proteolysis of Aldolase B Requires Ubiquitination

Earlier in this chapter, ubiquitinated aldolase B in liver was shown to contribute

to ubiquitin conjugates that are enriched in autophagic organelles during starvation-

induced autophagy. Ubiquitination was required for heat stress-induced delivery of

endogenous aldolase A to lysosomes ofE36 cells, suggesting that aldolase A was

degraded via ubiquitin-mediated autophagy. To examine whether aldolase B undergoes

ubiquitin-mediated autophagy like aldolase A, subcellular fractionation studies in the last

section were repeated with E36 and ts20 cells expressing epitope-tagged aldolase B

(RABM).

E36 and ts20 cells were transfected and selected for permanent expression of rat

aldolase B with the 9E10 myc epitope on its carboxyl terminus (RABM). The 9E10

epitope allowed efficient immunoprecipitation needed for degradation assays and











a.







52


;1~
I,


~.'
I.


Ph .


b.







0
(N1


I nS

'S




0


Figure 3-6: Transient Expression of RABM. a) E36 cells and b) ts20 cells transiently
expressing rat aldolase B with a carboxyl terminal myc tag (RABM) were processed for
immunofluorescence microscopy (Materials and Methods) and labeled with antibody
against aldolase B (ALDB) or the myc epitope (9E 10). Phase contrast (Ph) images for
corresponding fields are shown below each immunofluorescent micrograph. Scale bar =
50 4M.


9E10


4











unambiguous identification of the exogenous aldolase B, RABM. In Figure 3-6,

transient expression of RABM was easily detected in a small fraction of cells with

antibody to either the myc epitope (9E 10) or to native aldolase B (ALDB). Many

unlabeled cells indicated that E36 and ts20 cells do not express endogenous aldolase B.

Cell lines were isolated and screened for permanent RABM expression, and the highest

expressing lines for E36 and ts20 cells were designated E36AB and ts20AB, respectively

(Figs. 3-7 and 3-8).

After clonal selection all cells in a microscopic field were labeled for RABM in

permanent cell lines. Though most cells were brightly labeled, some were only dimly

labeled. Such labeling remained constant after multiple culture passages and for different

cell lines, suggesting that the variability was a trivial artifact of the immunofluorescence

protocol. According to immunofluorescence and western blot assays, different cell lines

had characteristic RABM levels that were maintained after multiple passages (data not

shown). Control experiments performed with cell lines expressing 5 to 10 fold

differences in RABM levels gave similar results. To facilitate detection, the highest

expressing lines (E36AB and ts20AB) were used extensively, and data are reported for

these lines. Immunofluorescent morphology indicated that RABM predominated in the

cytosol as shown by the presence of negatively labeled nuclei and vacuoles, providing

evidence that the recombinant protein demonstrated normal localization (Figs. 3-6, 3-7,

and 3-8).










E36


E36AB


W.. p '.


Figure 3-7: Permanent Expression of RABM in E36 Cells. Transiently transfected E36
cells were selected and screened for permanent RABM expression; the highest
expressing cell line (E36AB) and untransfected cells (E36) were processed for
immunofluorescent detection as described in Materials and Methods with anti-9E10 myc
epitope (9E10, upper panels); phase contrast for corresponding fields are shown (Phase,
lower panels). Scale bar =50 pM.


i'i. it










ts20


ts20AB


Figure 3-8: Permanent Expression of RABM in ts20 Cells. Transiently transfected ts20
cells were selected and screened for permanent RABM expression; the highest
expressing cell line (ts20AB) and untransfected cells (ts20) were processed for
immunofluorescent detection as described in Materials and Methods with anti-9E10 myc
epitope (9E 10, upper panels); phase contrast for corresponding fields are shown (Phase,
lower panels). Scale bar = 50 jtM.











E36 E36AB ts20 ts20AB
1 2 3 1 2 3 1 2 1 2 3


QQ
_g







0


Figure 3-9: Biochemical Detection of RABM Expression. Three confluent cultures (1,
2, 3) for each of the indicated cell lines was trypsinized, pelleted, suspended in 2 x
sample buffer, boiled, divided into two aliquots, run on duplicate SDS-PAGE gels,
western blotted, and duplicated blots were stained for aldolase B (upper panel) or the
9E10 myc epitope (lower panel); E36, original Chinese hamster lung cell line; ts20, an
E36-derived ts-mutant in ubiquitination; E36AB; an E36-derived cell line permanently
expressing RABM; ts20AB, a ts20-derived cell line permanently expressing RABM.
Arrow heads mark molecular weights (kD) for a doublet detected with anti-aldolase B.


-4 41.3
""40.0









-"40.0









On western blots of whole cells labeled with anti-aldolase B (Fig. 3-9), a single

faint 40 kD band was detected in untransfected cells (E36 and ts20), consistent with

cross-reactivity to aldolase A (Fig. 3-2b). Lysates of E36 and ts20 cells had aldolase

cleavage activity 35 to 40 fold higher for fructose 1,6-diphosphate than for fructose-1-

phosphate (data not shown). This difference for the two aldolase substrates is

characteristic for aldolase A, identifying this isoform as the prevalent endogenous

aldolase of E36 cells; note, aldolase B was not detected by immunofluorescence (Fig. 3-

6).

For RABM-expressing cells (E36AB and ts20AB), a closely spaced doublet of

bands was labeled with aldolase B antisera on western blots of whole cells (Fig. 3-9).

One band occurred at 40 kD coincident with endogenous aldolase A of untransfected

cells (E36 and ts20). A second strongly labeled band occurred at SDS-PAGE mobility

corresponding to 41.3 0.1 kD (mean SEM, n = 6). On duplicate blots labeled for the

C-terminal myc epitope of RABM (9E 10), long luminographic exposure times only

showed the 41.3 kD band (Fig.3-9). 41.3 kD matched the predicted molecular weight of

RABM, confirming that E36AB and ts20AB cells express full-length RABM.

If differences between immunoreactivities for hamster aldolase A (E36 cell

endogenous) and rat aldolase B (RABM) are similar to differences between titered

immunoreactivities (Fig. 3-2b) for purified rabbit aldolase A (Sigma) and purified human

aldolase B (Fig. 3-1), then immunoreactivities can be used to estimate RABM expression

relative to endogenous aldolase A. Endogenous aldolase A immunoreactivity was below








82
the linear range of western blot ECL assays in sample sizes subsaturating for aldolase B

detection. Immunodetection in the low range variably underestimated aldolase A by 3 to

>5 fold (data not shown), but allowed an upper limit for relative RABM expression to be

estimated. Estimates varied in the range of 0.2-2.4 RABM per endogenous aldolase A.

Though not precise, these overestimates indicated that RABM levels in permanent lines

were equal or less than the endogenous.

To determine whether RABM undergoes heat stress-induced autophagy, cultures

ofE36AB and ts20AB were incubated at control (CT) and heat stress (HS) temperatures

and fractionated as done for untransfected cells in the previous section titled, "Heat

Stress-Induced Autophagy of Aldolase A Requires Ubiquitination." Subcellular

distributions for activities of aldolase A and acid phosphatase were indistinguishable

between parental and RABM-expressing cell lines. The enzymatic activity for aldolase A

is -10 fold more than aldolase B (Penhoet and Rutter, 1975). Given this, aldolase B

enzymatic activity was too low to detect above endogenous aldolase expression in E36

cells. To overcome this problem, aldolase B immunoreactivity was followed on western

blots of subcellular fractions (Fig. 3-10).

Figure 3-1Oa compares the distribution of immunoreactivities for RABM

(Aldolase B) and an integral membrane protein of lysosomes (LAMP2b) in subcellular

fractions isolated from E36AB cells incubated at control temperatures (CT). Details of

the subcellular fractionation are described in Materials and Methods. When cell sheets

were scraped from culture dishes and pelleted, some soluble proteins leaked out of the

cells, as indicated by the presence of RABM (Aldolase B) in the supernatant (Sc, Fig. 3-















Figure 3-10: RABM Associated With Lysosomes Undergoes Ubiquitin-Mediated
Proteolysis. Confluent E36AB and ts20AB cell cultures were incubated for 8.5 total
hours at indicated conditions and then fractionated as described in Materials and
Methods; samples of fractions were separated by SDS-PAGE, western blotted and
labeled for distribution of lysosomal membranes on higher molecular weight half of blots
(upper panels) and RABM on lower half of blots (lower panels) using antibodies to an
integral membrane protein of lysosomes (LAMP2b) or to native aldolase B (Aldolase B);
a) E36AB cells incubated in CT (see below) conditions were used to show distributions
for lysosomal membranes and RABM which were comparable for all treatments within
the variability of ECL detection (Amersham), 1% total culture equivalent of fractions
was loaded per lane except HP (lane 6) which used 10%: Sc, supernatant from cells
scraped then pelleted at 1000 x g; Ho, homogenate of lysed cell pellet; LP & Lsu, pellet
& supernatant from 1000 x g of Ho; HP & Hsu, pellet & supernatant from 100,000 x g
of Lsu; b) HP fractions isolated from cells incubated under indicated conditions were
loaded for equal acid phosphatase activity to show relative content of full-length RABM
(Aldolase B, 41.3), proteolyzed RABM (Aldolase B, 40.0), and lysosomal membranes
(LAMP2b): Co, control temperature, in DMEM + 10% FBS (normal culturing
conditions); CT, control temperature in MEM medium (experimental control); HS, heat
stress in MEM medium; +CHQ, medium supplemented with 80 PM chloroquine.



Simplified Diagram of Fractions*:

Cell Sheet --> scrape & 1000 x g --> sup = Sc

pel, homogenize = Ho

1000 x g -+ sup = Lsu -> 100,000 x g -*> sup=
Hsu
4-pel = LP pel = HP
pel =LP pel =HP


*sup, supernatants; pel, pellets










a. E36AB CT
Sc Ho LP Lsu Hsu HP

tNI

135
1 2 3 4 5 6


-o- ~ w


b. HP Fractions
E36AB ts20AB


HS+
Co CT HS CHQ


Co CT HS


NOW


1 2 3 4 5 6 7


v --.41.3
-w 40.0


-.41.3
" 40.0









85
10a, lane 1). Membrane-bound organelles were retained in cells as indicated by the lack

of LAMP2b label in Sc.

The cell pellet was lysed, homogenized (Ho, Fig. 3-10a, lane 2), and used to

produce 1000 x g (low speed) pellet (LP) and supernatant (Lsu). Low speed pellets

generally contain nuclei, large cell fragments, and unbroken cells (Rickwood, 1992). LP

contained no detectable RABM, indicating that most cells were broken enough to lose

soluble proteins to Lsu (Fig. 3-10, lanes 3 and 4). Supporting this, LP contained about

15% of the aldolase A activity contained in Lsu (data not shown). The fact that some

aldolase A (Fig. 3-4a) but no RABM was detected in LP is probably due to the greater

affinity of aldolase A for pelletable cell components compared to aldolase B (Kusakabe,

et al., 1997). LAMP2b was approximately equally distributed in LP and Lsu, indicating

that about half the organelles (at least lysosomes) cofractionated with large cell

fragments or nuclei. In agreement with this, acid phosphatase activity was similarly

distributed between LP and Lsu (data not shown).

The Lsu was then used to produce 100,000 x g (high speed) pellet (HP) and

supernatant (Hsu). Such high speed centrifugations pellet all membrane-bound

organelles and leave soluble cytosolic (and leaked organellar) components in the

supernatant (Rickwood, 1992). Consistent with this, all the detectable lysosomal

membranes (Fig. 3-10a, LAMP2b) in Lsu (lane 4) were pelleted out of Hsu (lane 5). To

make RABM (Aldolase B) labeling in HP (Figure 3-10Oa, lane 6) comparable to that

loaded in lanes containing cytosol, 10 fold more HP equivalent was loaded.









86
On western blots of directly harvested whole cells (E36AB and ts20AB), aldolase

B immunoreactivity (RABM) primarily occurred at 41.3 kD (Fig. 3-9). In subcellular

fractions, a large proportion (>40%) of aldolase B immunoreactivity occurred at 40 kD

(Fig. 3-10a). This indicated that 41.3 kD RABM was proteolyzed to -40 kD size during

fractionation. Like RABM, the LAMP2b-reactive protein was also proteolyzed as

indicated by the presence of a smear below its band on western blots (Fig. 3-lOa,

especially visible in 10X loaded HP, lane 6). EDTA and storage on ice was used to

reduce protein degradation in subcellular fractionations but was insufficient to prevent

this presumably artifactual proteolysis. However, this result suggested that processing of

RABM from 41.3 kD to 40 kD could be used as an indicator of proteolysis.

Consistent for all treatments and fractions containing RABM (except HP), the

artifactual proteolysis was limited to processing 40-60% of the RABM (Fig. 3-10a, lanes

1, 2, 4, and 5). Only in HP fractions, processing of 41.3 kD RABM to 40 kD was

complete or nearly complete, such that aldolase B immunoreactivity collapsed from a

doublet to a single band at 40 kD (Fig. 3-1Oa, lane 6; Fig. 3-lOb, lanes 1, 2, 3, 5, and 6).

However, if lysosomal degradation was inhibited by chloroquine (E36AB, HS+CHQ) or

autophagic degradation blocked by the non-permissive ubiquitination of heat stressed

ts20 cells (ts20AB, HS) then the complete processing of RABM in HP fractions was

blocked, as indicated by the persistence of 41.3 kD RABM in a doublet (Fig. 3-lOb,

lanes 4 and 7). These data indicated that RABM proteolysis occurred in lysosomes

(CHQ-sensitive) and required ubiquitination (blocked in HS ts20AB). Whether greater










proteolysis of RABM seen in HP occurred in cells or during fractionation was not

determined. With the data from the last section, the results suggest that RABM

(aldolase B), like endogenous aldolase A, utilizes lysosomal degradation that requires

ubiquitination in E36 cells.

To demonstrate the presence of ubiquitinated aldolase B in E36 cells expressing

RABM, E36AB and ts20AB cell cultures were incubated at the different conditions used

for subcellular fractionation studies above. Then RABM protein was isolated using

9E10-specific immunoprecipitation and separation on SDS-PAGE (Materials and

Methods). Gels were western blotted and detected with native (N) and denatured (D)

aldolase B antisera. Since antisera for denatured aldolase B were most sensitive and

specific for ubiquitinated aldolase B on western blots (Fig. 3-2a and 3-3), these

antibodies were used to probe for ubiquitinated aldolase B (Fig. 3-11 a, upper panel). A

major stable ubiquitin-aldolase B conjugate occurred at 68 kD consistent with Ub68

found in rat liver (Fig. 1-3 and 3-3) and in Fao hepatoma cells (Fig. 1-4). This confirmed

that E36AB and ts20AB cells contained ubiquitinated aldolase B, including Ub68.

Similar levels of Ub68 were detected in all conditions, including heat stressed ts20 cells

(ts20AB, HS MEM) in which ubiquitination is inhibited. Unchanged ubiquitinated

protein under conditions of inhibited ubiquitination appears contradictory. Though

greatly inhibited, a low level of ubiquitination continues in heat stressed ts20 cells, but

this low level is insufficient to mediate cellular processes (Hischberg and Marcus, 1982;

Kulka, et al., 1988; Gropper, et al., 1991). The results here suggest that low levels of

ubiquitination were sufficient to maintain multiubiquitinated intermediates of aldolase B






















Figure 3-11: Ubiquitinated Aldolase B Occurs in E36 and ts20 Cells Expressing RABM.
Replicate sets of E36AB and ts20AB cultures were treated with indicated media
(DMEM +FBS or MEM) and temperatures (CT or HS), harvested, and
immunoprecipitated with 9E 10 antibody; the immunoprecipitate was pelleted (P) from
the lysate and proteins remaining in the supernatant were precipitated with
trichloroacetic acid (S); P and S samples were boiled in 2 x sample buffer, split in equal
aliquots, and duplicate gels run on SDS-PAGE; a) Western blots to detect RABM
immunoreactivity (Aldolase B) were made from one gel and upper and lower portions of
blots were immunodetected with anti-denatured (upper panel, D) and anti-native (lower
panel, N) aldolase B, respectively; b) the duplicate gel was Coomassie stained, lane
numbers at the top of the Coomassie gel correspond to identical samples in lanes
numbered at the bottom of the blots in (a.); MW, molecular weight markers; molecular
weights (kD) are indicated at right.








E36ABts20AB


CT
DMEM+FBS MEM
P S P S


HS CT
MEM DMEM+FBS MEM
P S P S P S


*f lr '-


HS
MEM
P S


9"'


N q .rA


4 *. 68


I


0o


E


S^W^


-.41.3
-40.0


1 2 3 4 5 6 7 8 9 10 11 12
b. MW 1 2 3 4 5 6 7 8 9 10 11 12 MW


-466.2

455.0
442.7
440.0


4 31.0


4 21.5


E36AB


Dt









90
(Ub68 probably contains 4 ubiquitins), but were insufficient to mediate the proteolysis of

RABM detected in HP fractions (Fig. 3-10b, lane 7). The occurrence of Ub68 in

different samples from different cell types (Figs. 1-3, 1-4, and 3-11) supports it as a

stable basal intermediate that probably requires more ubiquitination to facilitate

proteolysis. Alternatively, heat stress-induced ubiquitination could operate on the

machinery of stress-induced degradation. Further experiments are needed to distinguish

these alternatives.

As detected with antiserum to native aldolase B (N), immunoprecipitation with

the 9E10 epitope (specific for RABM) effectively pelleted all detectable 41.3 kD RABM

protein and more than half of a dim-labeled 40 kD protein, presumably endogenous

aldolase A (Fig. 3-1 la, lower panel). The 40 kD aldolase A was shown to lack 9E10

immunoreactivity (Fig. 3-9), indicating that RABM and endogenous aldolase A occur as

a complex in E36AB and ts20AB cells. This is consistent with the known tetrameric

structure of all FDP aldolase isozymes, wherein different subunits randomly and stably

oligomerize during synthesis (reviewed in Chapter 1). Ubiquitinated forms of aldolase B

were also removed from lysates by 9E10 immunoprecipitation (Fig. 3-1 la, upper panel),

suggesting that ubiquitinated RABM retained its C-terminal epitope tag or retained

associations with unmodified 9E10-immuno-reactive RABM subunits. If ubiquitinated

RABM is not associated with other aldolase subunits, then this would provide evidence

that ubiquitination might disassemble quaternary structure of aldolase B perhaps as an

early step in the degradative pathway. However, this possibility was not pursued here.










Experimental
Treatment

A. Control]


Association
With
Organelles


Early
Intermediates


Limited
Lysosomal
Proteolysis


Late
Intermediates


Sbasal Aldolase A 40 kD RABM
(trace, 41.3kDRABM)


B. IHeat Stress Aldolase A 40 kD RABM
(trace, 41.3 kD RABM)

C. HeatStress 1 Aldolase A kD_
+chloroquine : 41.3 kD RABM 40 kD RABM



D. Heat Stress basal Aldolase A 40 kD RABM
ts20 mutant {^ _41.3 kDRABM



Figure 3-12: Summary of Association and Limited Proteolysis of RABM and
Endogenous Aldolase A in Pelleted Organelles of E36 Cells. Each pathway corresponds
to experimental treatment described in boxes at left and correspond to the following
abbreviations used above: A) E36AB or ts20AB, CT; B) E36AB, HS; C) E36AB, HS
+ CHQ; D) ts20AB, HS. Since aldolase A was detected by enzymatic activity and is
inactivated by limited proteolysis, no late intermediates of Aldolase A are shown. Since
RABM detected on western blots shifts from 41.3 kD to 40 kD forms by limited
proteolysis, these forms are listed for early and late intermediates, respectively. Weight
of white vertical arrows indicate relative increases in detected levels of aldolase A and
RABM pelleted with organelles. Weight of black horizontal arrows indicate relative
rates proposed for processes listed in the heading.


Figure 3-12 summarizes the results of subcellular fractionation studies for

endogenous aldolase A and RABM expressed in E36 cells. Aldolase A activity is very

sensitive to proteolytic inactivation and loses 98% of its activity upon limited proteolysis

(Penhoet and Rutter, 1975; Horecker, et al., 1985). Since this made proteolyzed









92
aldolase A undetectable in the background of active aldolase A from E36 cells, aldolase

A activity was used to demonstrate association with organelles but not for detecting

proteolyzed intermediates of degradation. RABM was detected by western blotting with

antiserum to native aldolase B, allowing detection of limited proteolysis (41.3 kD --+ 40

kD) products referred to here as "late intermediates" (Fig. 3-12, last column). Smaller

molecular weight intermediates of proteolysis were not detected, because they are not

recognized by anti-native aldolase B and probably are more rapidly degraded than 40 kD

aldolase B (Reznick, et al., 1985; Horecker, et al., 1985).

Under control conditions (Fig. 3-12, A), basal levels of aldolase A, 40 kD

RABM, and a trace of 41.3 kD RABM were detected in pelletable organelles. Heat

stress (Fig. 3-12, B) caused a partial increase in aldolase A activity with little apparent

change in RABM forms. However, a partial increase in trace levels of 41.3 kD RABM

were likely to be missed, because they were below the threshold for optimal Enhanced

Chemiluminescent detection (Amersham). Consistent with reaching the threshold for

detection, chloroquine inhibition of limited proteolysis caused a sudden signal increase in

41.3 kD RABM (Fig. 3-12, C). Chloroquine also caused an even more aldolase A to

accumulate. The results indicated that lysosomal proteolysis mediates loss of aldolase A

and aldolase B (RABM) associated with organelles, demonstrating sequestration of these

proteins into lysosomes.

When ubiquitination was inhibited by the ts20 mutation (Fig. 3-12, D) different

results were obtained for aldolase A and aldolase B (RABM). Consistent with




Full Text
CELLULAR MECHANISMS FOR
THE REGULATED DEGRADATION OF ALDOLASE B
By
Peter P. Susan
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
1998

TABLE OF CONTENTS
page
ABSTRACT vi
CHAPTERS
1 INTRODUCTION 1
General Concepts of Protein Degradation 1
Background for Fructose 1,6-Diphosphate Aldolase B 3
Mechanisms for Degradation Aldolase B 8
Hypothesis for Stress-Induced Degradation of Aldolase B 28
General Strategy 30
2 MATERIALS AND METHODS 33
Cell Lines and Culturing 33
Plasmid Vector Construction and Mutagenesis 35
ii

Expressing Epitope-Tagged Aldolase B in Cell Lines 41
Immunofluorescence 44
Antibodies 44
Viability Assays 48
Subcellular Fractionation 49
Enzyme Assays 52
Protein Analysis 53
Stress-Induction of Protein Degradation 55
Protein Degradation 57
3 UBIQUITINATION MEDIATES LYSOSOMAL PROTEOLYSIS OF
ALDOLASE B 59
Introduction 59
In Vivo Multiubiquitination of Aldolase B 60
Multiubiquitinated Aldolase B is Denatured and Enriched in Lysosomes 68
Heat Stress-Induced Delivery of Aldolase A to Lysosomes Requires
Ubiquitination 69
Heat Stress-Induced Proteolysis of Aldolase B Requires Ubiquitination 75
iii

Ubiquitin-Mediated Autophagic Degradation Occurs in E36AB Cells
93
4 TEMPERATURE MODULATES AUTOPHAGY AND CYTOSOLIC
PROTEOLYSIS OF ALDOLASE B 101
Introductions 101
Ubiquitin-Independent Cytosolic Proteolysis of Aldolase B 102
Temperature-Dependent Cytosolic Proteolysis in Fao Cells 106
Starvation-Induced Autophagic Degradation of Aldolase B in Fao Cells... 110
Temperature-Dependent Autophagy and Cytosolic Proteolysis 118
A Model For the Degradation of Aldolase B 123
5 SIGNAL-MEDIATED DEGRADATION OF ALDOLASE B 127
Introduction 127
Transient Expression of RABM Mutations in Putative Lysosome
Targeting Signals 130
Starvation Induces Autophagic Degradation in HuH7 Cells 139
Transient Expression Does Not Affect Starvation-Induced Degradation
of RABM 140
Site-Directed Mutations Did Not Affect Wildtype Activity of RABM 142
Glutamine Residue #111 is Required for Starvation-Induced Degradation
of Aldolase B 143
Glutamine #111 Specifically Mediates Starvation-Induced Degradation of
Aldolase B 146
6 SUMMARY AND CONCLUSIONS 151
IV

Introduction
151
Autophagy and Ubiquitination 151
Clues from Temperature-Dependent Cytosolic Proteolysis and
Lysosomal Degradation 154
Signal-Mediated Targeting 157
Present and Future Contributions to the Field of Protein
Turnover 153
REFERENCES 164
BIOGRAPHICAL SKETCH 176
v

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
CELLULAR MECHANISMS FOR
THE REGULATED DEGRADATION OF ALDOLASE B
By
Peter P. Susan
August 1998
Chairman: William A. Dunn, Jr.
Major Department: Anatomy and Cell Biology
Stress-induced degradation of abundant long-lived cytosolic housekeeping
proteins was examined using liver aldolase B as a model protein. Heat stress increases
ubiquitination that mediates autophagic degradation of long-lived proteins in E36
Chinese hamster cells. During starvation, major multiubiquitinated proteins (e.g., Ub68)
increased in lysosomes (rat liver and Fao hepatoma cells) and were antigenically
characterized as aldolase B-ubiquitin conjugates. Compared with controls, heat stress
increased endogenous aldolase A activity in lysosomes of E36 cells by >twofold. Heat
stress was non-permissive for ubiquitination in E36-derived ts20 mutant cells and failed
to increase aldolase activity in ts20 lysosomes. Myc-tagged aldolase B (RABM)
expressed in E36 cells underwent limited proteolytic processing in lysosomes that failed
vi

to occur in heat stressed ts20 cells. The results suggested that during stress (starvation
or heat), aldolase A and aldolase B can undergo ubiquitin-mediated autophagic
degradation. Long-lived protein degradation was a continuous function of temperature,
indicating heat stress-induced rates were due to thermodynamic stimulation of chemical
reactivity. Lysosomal inhibitors distinguished proteolysis in lysosomes from that in
cytosol. Complete autophagic degradation to amino acids in lysosomes was highly
temperature-dependent compared to a relatively constant rate in cytosol. HA-tagged
human aldolase B (HAHAB) in Fao cells and RABM in E36 cells underwent proteolysis
in cytosol that had temperature-dependence paralleling complete degradation of proteins
in lysosomes. Lysosomal degradation was ubiquitin-dependent (blocked in heat stress
ts20 cells), but cytosolic proteolysis of RABM was not. Results suggest a possibly
shared temperature-dependent cytosolic mechanism that limits rates for partial cytosolic
proteolysis and complete lysosomal degradation of long-lived proteins. Three peptide
motifs for signal-mediated targeting to lysosomes during starvation occur in aldolase B.
These were mutated in RABM. Starvation-induced degradation of mutant and wildtype
RABM expressed in HuH7 human hepatoma cells were measured. Starvation-induced
degradation of RABM (aldolase B) specifically required a glutamine at residue #111
suggesting that the corresponding peptide motif, IKLDQ, is a targeting signal
functionally demonstrated in living cells. Evidence was provided for three previously
unknown mechanisms for stress-regulated degradation of aldolase B: (1) ubiquitin-

mediated autophagic degradation in lysosomes, (2) temperature-dependent cytosolic
proteolysis during heat stress, and (3) signal-mediated degradation during starvation.

CHAPTER 1:
INTRODUCTION
General Concepts of Protein Degradation
In cells, different proteins have different functions and occur at different levels as
needed. The functional activities and locations of proteins are regulated to integrate with
each other, maximizing survival. Proteins can be regulated by a variety of mechanisms,
but available concentration of each protein fundamentally determines maximal function
(Doherty and Mayer, 1992). Cells adapt to environmental change by altering amounts of
different proteins. Some increase, others decrease, and the rest are constant (Doherty
and Mayer, 1992). Such adaptation of different proteins requires preferential
mechanisms for regulating synthetic or degradative rates in response to environmental
change.
Under constant conditions, protein synthesis is independent (zero order
relationship) of the protein concentration, but degradation is directly proportional (first
order relationship) to protein concentration (Doherty and Mayer, 1992). Synthesis
increases protein concentration, causing degradation to increase until synthetic and
degradative rates are equal. In this way, a balance between synthesis and degradation
determines the available concentration of a protein. If environmental conditions change,
then cells can adapt protein concentrations by modulating synthesis or degradation
1

2
(Olson, et al., 1992; Mortimore, 1987). This study examines mechanisms of protein
degradation that respond to environmental changes.
Continual synthesis and degradation results in constant turnover of proteins
which can be described by either the fractional degradation rate or the half-life of the
protein. The fractional degradation rate of a protein (degradative rate constant), is
defined as the fraction of the initial protein degraded in a given time. The k¿ is calculated
from measurements of labeled protein lost per time. Half-life, Xy„ is defined as the time
for turnover of half the protein. Under equilibrium conditions, and t% are constant and
directly related to each other by = ln(2)/ kd, allowing calculation of ty2 from
experimentally determined kd (Doherty and Mayer, 1992).
Proteins are categorized as short-lived for XVl < 1 h or long-lived for XVl >1 h. For
short-lived proteins, protein concentrations respond more to changes in synthesis (Olsen,
et al., 1992). For long-lived proteins, protein concentrations respond more to changes in
degradation (Olsen, et al., 1992; Mortimore and Poso, 1987). Detailed reasoning for
this is described elsewhere (Doherty and Mayer, 1992). Many short-lived proteins utilize
a well-characterized mechanism for degradation by a cytosolic protease complex called
the proteasome (Rock, et al., 1994; Ciechanover and Schwartz, 1994; Hochstrasser,
1992). Relative to short-lived proteins, mechanisms for the degradation of long-lived
proteins are poorly characterized. This dissertation examines mechanisms for stress-
inducible degradation of a long-lived cytosolic enzyme, fructose 1,6-diphosphate
aldolase (see next section).

3
Mechanisms for degradation of proteins become enhanced during environmental
stress. Increased temperature (Bates, et al., 1982; Hough and Reichsteiner, 1984) or
nutrient deprivation (Mortimore and Poso, 1987) are known to increase the degradation
of long-lived proteins. Stress-induced degradative mechanisms are of special interest,
because they mediate regulated changes and themselves must be regulated (Mortimore,
et al., 1987; Mortimore and Poso, 1987; Olson, et al., 1990). Any mechanism that can
be triggered by environmental stress lends itself to experimental manipulation. Such a
mechanism can be modulated simply by changing experimental conditions. Furthermore,
mechanisms required for a stress-induced degradation can be shown to be specific by
lack of effect on basal mechanisms. For example, cells respond to starvation by
increasing the degradation of long-lived proteins. 3-Methyladenine is a drug that
specifically blocks the enhanced degradation without affecting basal degradation. 3-
Methyladenine is a potent inhibitor of autophagy, a mechanism for delivering proteins to
lysosomes for degradation. Such results provide evidence that autophagy plays a role in
enhanced degradation but not basal degradation of proteins.
Background for Fructose 1.6-Diphosphate Aldolase B
In the next section, I discuss potential mechanisms for the degradation of the
liver isoform of fructose 1,6-diphosphate (FDP) aldolase, called aldolase B. Molecular
mechanisms for the degradation of aldolase B have not been examined, but preliminary
examination indicated potential roles for ubiquitination and signal-mediated delivery to
lysosomes. Before examining the degradation of aldolase B, this section serves to

4
familiarize the reader with aldolase B in relation to other aldolase isoforms. There are
two major classes (I and II) of FDP aldolase that have no sequence homology
(Alefounder et al., 1989) and utilize different catalytic mechanisms (Rutter et al., 1966).
Class II aldolases are only found in micoorganisms and are considered no further here,
but class I aldolases are represented in all taxons (Rutter et al., 1966). Microorganisms
variably lack class I aldolase. For example, some strains of E.coli contain class I
aldolase (Alefounder et al., 1989), whereas strain JM83 lacks class I activity (Sakakibara
et al., 1989). Protozoa and muticellular eukaryotes contain class I aldolases.
The FDP aldolase isoforms of mammals are the best characterized. Most studies
of FDP aldolase degradation examine mammalian isoforms. Mammalian aldolase
isoforms are synthesized from three separately regulated genes coding different proteins.
Muscle, liver, and brain each express one predominant isoform designated A, B, and C,
respectively (Rutter et al., 1966). As such, liver aldolase and aldolase B are synonyms.
Likewise, aldolase A is also called muscle aldolase, and aldolase C is referred to as brain
aldolase. Most tissues, including embryonic, contain combinations of aldolase A and C,
but aldolase B appears to be exclusively expressed in liver and kidney cells (Rutter et al.,
1966).
Aldolase classes and isoforms are distinguished by clear differences in their
enzymatic properties. For example, class I aldolase like that in mammals is totally
functional in the presence of EDTA, but class II enzymes of E. coli and other microbes
are completely inhibited. The three mammalian isoforms (A, B, and C) can be

5
distinquished by differences in specific activity, sensitivity to carboxypeptidase A, and
kinetics (Vmaxand Km) for different substrates (Rutter et al., 1966).
Aldolases A, B, and C also are characterized by distinct native epitopes. Thus,
antibody to one poorly recognizes the others. However, an antibody against a specific
isoform for one animal will similarly recognize the same isoform of a different species, at
least amongst mammals (Penhoet and Rutter, 1975). Native and denatured epitopes of
aldolase B have also been shown to be distinct from each other (Reznick et al., 1985).
Chemical denaturation of aldolase B before immunization resulted in antibody that failed
to immunoprecipitate native enzyme activity but could precipitate degradative fragments
of aldolase B. Since aldolase has stable structure that spontaneously refolds into native
conformations, only fragments sufficiently denatured by degradation were detected by
antibody against the denatured aldolase B (Reznick et al., 1985). Anti-native aldolase B
had converse immunoreactivity. Thus, it was proposed that three-dimensional
conformation is important for antibody recognition of native surface epitopes, whereas
the native structure buries and masks denatured epitopes (Reznick et al., 1985).
The three dimensional structure of all class I aldolase isoforms is conserved from
bacteria to humans (Alefounder, et al., 1989). Secondary and tertiary structures of
aldolase are very stable. This is true at the quaternary level, too. Aldolase occurs as
very stable tetramers that do not undergo subunit exchange after synthesis (Lebherz,
1975; Lebherz, 1972). Different aldolase isoforms co-synthesized in the same cell
randomly associate into stable heterotetramers. Thus, immunoprecipitation with

6
antibody against one isoform specifically precipitates antigenically unrelated isoforms in
the same tetramer. However, surface charge and pi on different isoforms varies. Thus,
isozymes containing different ratios of two isoforms (e.g. A4, A3B, A2B2, AB3, or B4)
can be separated by isoelectric focusing (Lebherz, 1972; Penhoet and Rutter, 1975).
Comparison of X-ray crystallographic results shows that secondary and tertiary structure
between muscle, liver, and Drosophila aldolases are very close (Berthiaume et al., 1993).
Aldolase isoforms vary in their capacity to bind actin cytoskeletons. In the only
paper to measure cytoskeletal association of all three isoforms (A, B, and C),
investigators claimed that different purified isoforms of aldolase had tissue-specific
affinity for cytoskeletal preparations isolated from different tissues (Kusakabe et al.,
1997). After mixing crude cytoskeletons with a known amount of purified aldolase, they
pelleted the mixture and then only measured unbound activities in supernatants. They
failed to show removal of endogenous aldolase from cytoskeletal preparations, so
measurements could be contaminated and include competitive effects. However, their
results were consistent with other investigators in that relative tightness of binding to
actin cytoskeleton is greatest to least, aldolase A, aldolase B, then aldolase C (Clarke, et
al., 1982; O'Reilly and Clarke, 1993).
Four “isotype specific” sequences contain most of the variation and the carboxyl
terminus has the greatest diversity (Marchand et al., 1988; Paolella et al., 1984;
Rottmann et al., 1984). The carboxyl terminus is important in determining isoform-
specific catalytic properties (Berthiaume et al., 1993; Gamblin et al., 1991; Penhoet and

7
Rutter, 1975). Aldolase B has the lowest specific activity amongst aldolase isoforms and
is least sensitive to proteolytic alterations in this region. It can lose up to four C-terminal
residues without affect its enzymatic activity (Berthiaume et al., 1993; Horecker et al.,
1985). Even with 10 to 20 residues removed by carboxypeptidase, aldolase B retains
almost half its activity (Penhoet and Rutter, 1975). However, aldolase A absolutely
requires a carboxyl terminal tyrosine at residue 364 (Y364) for activity that is about 20
times greater than aldolase B, and when aldolase A loses its C-terminus the remaining
activity resembles that of aldolase B (Takahashi et al., 1989; Gamblin et al., 1991).
These results indicate that alterations in the carboxyl terminus of aldolase B (such as
epitope tagging) are less likely to affect its properties than other aldolase isoforms.
All fructose 1,6-diphosphate aldolase enzymes catalyze a reversible reaction
essential for glycolysis and gluconeogenesis. Aldolase B is the liver form of this enzyme
expressed to the exclusion of other forms of aldolase in normal hepatocytes (Asaka, et
al., 1983). Since liver is the only organ known to export glucose (Stein and Arias, 1976;
Stryer, 1988), aldolase B performs gluconeogenesis for the entire body. Liver also
provides amino acids during starvation and in three days can lose nearly half its weight
(and protein content), a faster loss than other tissues (Wing et al., 1991). In this regard,
aldolase B is an example of an abundant cytosolic protein that undergoes enhanced
degradation during starvation which yields amino acids for export to other organs. Liver
amino acids can also be converted to glucose or ketone bodies to provide energy sources
during starvation. Abundant long-lived liver enzymes that mediate

8
glycolysis/gluconeogenesis, like aldolase B and glyceraldehyde phosphate dehydrogenase
(GAPDH) are poised between two mutually exclusive functions: catalyzing carbohydrate
metabolism and providing amino acids for protein biosynthesis or energy metabolism.
Liver and kidney are the only tissues having predominant aldolase B expression
(Penhoet and Rutter, 1975). Both organs demonstrate enhanced degradation of proteins
during amino acid starvation (Olsen, et al. 1990). Liver and kidney also receive the
largest fraction of the body’s basal blood flow, 27% and 22%, respectively, followed by
15% for muscle and 14% for brain (Guyton, 1979). This is consistent with involvement
of the two former organs in regulating serum components and contribution of aldolase B
to serum glucose and amino acids during starvation. Among aldolase isoforms, aldolase
B contributes a greater role in carbohydrate and protein metabolism that is not limited to
local cells and tissues, but extends to the entire body.
Mechanisms for Degradation Aldolase B
Inactivation by Limited Proteolysis
Alteration of aldolase A and B’s carboxyl termini was proposed to down-regulate
activity (Pontremoli et al., 1982; Pontremoli et al., 1979). During starvation, aldolase B
activity is lost from liver faster than loss of immunoreactivity. Thus, investigators
suggested that starvation-induced inactivations precede total degradation of aldolase A
and B, providing more rapid down-regulation of activity (Pontremoli et al., 1979).
Inactivation happens by limited C-terminal cleavage that can maintain native

immunoreactivity and barely affect mobility on SDS-PAGE. One group of investigators
proposed phosphorylation near the C-terminus of aldolase as an inactivating mechanism,
but this was only demonstrated in vitro (Sygusch et al., 1990). More likely inactivation
occurs by limited proteolysis which would have a much more profound impact on
aldolase A activity than on aldolase B activity (previous section, last paragraph).
The best characterized mechanism for aldolase inactivation is limited proteolysis
by a dipeptidyl (two residues per cleavage) carboxypeptidase on lysosomes (Pontremoli
and Melloni, 1986; Horecker et al., 1985). The peptidase, cathepsin M, was defined as
a cathepsin B or L-like activity associated with the cytosolic surface of lysosomal
membranes. During starvation, a lysosomal matrix cathepsin B/L associates with
lysosomal membranes, acquires activity at neutral pH, and becomes exposed to the
cytosolic compartment as cathepsin M (Pontremoli et al., 1984; Pontremoli et al., 1982).
Specific cleavage sites have been characterized in vitro (Horecker et al., 1985).
Starvation-induced in vivo loss of liver aldolase specific activity correlated with
loss of carboxyl terminal tyrosine residues which was estimated by isolating aldolase B
and measuring lost tyrosine content in an acid soluble peptide released from the C-
terminus with subtilisin (Pontremoli et al., 1982). According to such experiments,
inactivated aldolase B constitutes about 40% of the aldolase in liver after 60 hours of
starvation. Most of the inactivated aldolase B must occur in cytosol, because only a
small fraction of total aldolase (about 10%) is associated with pelletable fractions from
liver (Kominami et al., 1983; Kopitz et al., 1990). Moreover, intralysosomal

10
degradation of aldolase is rapid (see below), precluding accumulation of an inactivated
form in such organelles. The results are consistent with inactivation occurring in the
cytosolic compartment, albeit by an activity associated with the cytosolic surface of
lysosomes.
In Vitro Denaturation of Aldolase and Need for In Vivo Mechanism
Except for 20 “loose” amino acid residues at the carboxyl terminus, the stability
of aldolase structure resists proteolysis and requires denaturation for rapid in vitro
proteolysis to proceed. In optimized conditions with cathepsin D, only about 20 amino
acids of aldolase A can be digested from its carboxyl terminus (Oflfermann et al., 1983).
In vitro proteolysis with either meprin (a metalloproteinase) or a mixture of lysosomal
proteases produces only a slight increase in SDS-PAGE mobility, and the remaining part
of aldolase A has a thermal stability identical to the native enzyme (Bond and Offermann,
1981). Purified aldolase B digested with a lysosomal extract also only undergoes
limited proteolysis, losing some but not all its activity (Chappel et al., 1978). However,
denaturing pretreatment with disulfides like glutathione (Offermann et al., 1983) or
cystine (Bond and Offermann, 1981) permits extensive proteolysis to occur. Given
this, there must be a “denaturing” mechanism in vivo to allow degradative turnover of
aldolase to occur. Interestingly, aldolase B sequestered in vivo and isolated with
lysosomes is susceptible to more extensive in vitro proteolysis in the lysosomal
preparations (Kominami, et al., 1983; Ueno and Kominami, 1991). Apparently,

11
aldolase B becomes sensitized to proteolysis by a mechanism in cytosol before
sequestration or in intact lysosomes after sequestration.
After loading aldolase A into endosomes at 19°C, temperature can be raised to
37°C allowing rapid fusion of endosomes with lysosomes. Thus, intralysosomal
degradation can be measured. By this method, native or variously denatured and
inactivated aldolases all degrade rapidly with similar rates (ty2 <10 min). Since its t* is
normally many hours in cytosol, sequestration appears rate limiting for lysosomal
degradation of aldolase (Bond and Aronson, 1983). The results of the endocytic loading
experiments indicate that a mechanism for denaturing and sensitizing aldolase to
proteolytic attack can occur in lysosomes or other organelles of the endosomal pathway.
Thus, a cytosolic denaturing mechanism is not necessary for intralysosomal degradation
of aldolase, but a role in delivery of aldolase to lysosomes cannot be excluded.
The tetrameric structure of aldolase is well established (Lebherz, 1972). This
quaternary structure seems important for aldolase stability. Recently, Beemink and
Tolan have indentified specific amino acids that mediate subunit interaction between
aldolase monomers (Beemink and Tolan, 1996). Significantly, a mutant with only two
amino acid changes retains enzymatic activity but exists as monomers. These monomers
(and dimers created with single amino acid mutations) are more sensitive to chemical or
thermal inactivation, indicating “looser” structure. Thus, tetrameric association
improves structural stability.

12
Lysosomes are acidic inside (pH ~5), and reversible in vitro dissociation of
aldolase into monomers occurs at pH < 6.0 (Beemink and Tolan, 1996). Acidic pH
affecting adolase structure is also indicated by reduced enzymatic activity. Thus,
intralysosomal pH would have a denaturing effect that could permit lysosomal
proteolysis. However, other investigators incubated aldolase B with crude lysosomal
hydrolases at acidic pH and failed to get significant proteolysis (Chappel et al., 1978).
Apparently, low pH is insufficient to permit further proteolytic attack, and aldolase
denaturation must require other factors. Consistent with this, lysosomes purified from
liver contain detectable aldolase B which is susceptible to proteolysis when the intact
lysosomes are incubated in vitro at pH < 5.5 (Kominami, et al., 1983; Ueno and
Kominami, 1991). The endocytic loading experiments described above indicate
lysosomes (or an endocytic compartment) must contain denaturing factors, but this does
not exclude the possibility of a cytosolic denaturation of aldolase B before delivery to
lysosomes.
Aldolase A has been radiolabeled, inactivated and denatured, then microinjected
into cultured cells (Hopgood et al., 1988; Knowles et al., 1989). The procedure delivers
the enzyme into cytosol where it normally resides. As with endocytic loading,
degradation rates for aldolase were similar whether the enzyme was native, inactivated,
or denatured. Denaturation of aldolase is not rate limiting for degradative steps before
lysosomes as well as within them. Degradation of aldolase microinjected into cytosol
matched expected turnover for aldolase (b/2 = 30 hours) which was much slower than for

13
aldolase loaded into lysosomes (t-/2 < 10 minutes). Assuming that degradation occurs
within lysosomes, this suggest that sequestration of aldolase is rate-limiting for its
turnover (Bond and Aronson, 1983; Bond and Offermann, 1981; Hopgood et al., 1988;
Knowles et al., 1989).
Though in vitro studies indicate denaturation of aldolase structure is necessary
for proteolysis, in vivo denaturation is not rate-limiting for delivery to or degradation
within lysosomes. These data support a model in which aldolase delivery to lysosomes is
rate limiting followed by rapid intralysosomal proteolysis which would need a faster
denaturing mechanism. Lysosomal acidity might facilitate denaturation of aldolase, but
acidity alone is insufficient for sensitizing stable aldolase structure to attack by acid
hydrolases. The above data do not exclude a cytosolic denaturing mechanism for
aldolase, but indicate that such a mechanism is not rate limiting and not necessary for
intralysosomal proteolysis. The next two sections review mechanisms for the delivery of
cytosolic proteins to the lysosomal lumen, a process that appears rate-limiting for
aldolase degradation.
Autophagv
Autophagy is the sequestration of cytoplasm into vesicles for intralysosomal
degradation and is the only mechanism proposed for the complete degradative turnover
of aldolase. There are two forms of autophagy: macroautophagy and microautophagy.
Commonly, investigators use the term “autophagy” to mean macroautophagy which is

14
the better characterized form. Likewise, “autophagy” used here refers to
macroautophagy, and reference to “microautophagy” will be explicit.
Autophagy (macroautophagy) begins with a ribosome-free portion of
endoplasmic reticulum engulfing a portion of cytoplasm. Autophagy non-selectively
sequesters cytosol and organelles into distinct autophagic vacuoles. The autophagic
vacuoles mature including a process of acidification. Finally, mature autophagic
vacuoles fuse with lysosomes producing autolysosomes in which degradation occurs
(Dunn, 1990; Dunn, 1990). Enhanced autophagy is initiated by amino acid starvation
and is also regulated by hormones (Hendil et al., 1990; Seglen and Bohley, 1992). In the
model of Figure 1-1, non-selective autophagy is represented by the upper pathway in the
diagram. The lower pathway of Figure 1-1 (Receptor-Mediated Targeting) is discussed
in the next section.
Microautophagy seems simpler than macroautophagy. During microautophagy
the lysosomal membrane itself invaginates, extending a finger of cytosol into the
lysosome. This protrusion pinches off producing an intralysosomal vesicle that gets
degraded with its cytosolic content. Apparently, microautophagy can occur in vitro, but
the complexity of macroautophagy has not been reconstitiuted (Seglen and Bohley,
1992). Unlike macroautophagy which has discrete autophagic vacuoles, microautophagy
fails to produce separable organellar compartments. Thus, microautophagy requires
time-consuming electron microscopy to demonstrate its exsistence and remains poorly
characterized (Seglen and Bohley, 1992).

15
Ribosome-Free
X s'"
Endoplasmic
‘9m
wrnmmmmmm
wmmmm
(íetramer)
Denature
iliiiiiii
Native
Subunit
LGP96
Unknown
Factor
Figure 1-1: Mechanisms for Stress-Induced Degradation of Cytosolic Proteins in
Lvsosomes. Autophagy (upper pathway) and receptor-mediated targeting (lower
pathway) were proposed for stress-induced delivery of cytosolic proteins to lysosomes
for degradation; the arbitrary cytosolic protein is shown as a tetramer (aldolase B occurs
as a tetramer); components of the pathways are labeled on the diagram; processes are
labeled by boxed numbers: 1, association with or engulfment by autophagic membranes;
2, sequestration into double-membrane bound autophagic vacuole; 3, maturation of
autophagic vacuole (acidification and acquisition of lysosomal hydrolases); 4, proteolysis
into polypeptide fragments; 5, complete degradation to amino acids; 6, disassembly and
denaturation of structure by an unknown factor; 7, association with a receptor complex
on the lysosomal surface; 8, translocation across the lysosomal membrane.
Selective mechanisms of autophagy exist. Methylotrophic yeast use a selective
mechanism of autophagy to degrade peroxisomes when switched from methanol to a
different carbon source, and electron microscopic morphology shows a mechanism

16
topologically identical to microautophagy (Tuttle et al., 1993). Occurrence of selective
microautophagy in higher organisms has not been demonstrated, and a role for
microautophagy in degradation of aldolase has not been studied. If selective autophagy
does occur for aldolase, then a receptor-mediated complex would be required for
selectivity. Such a receptor complex could form on the lysosomal surface (Fig. 1-1,
lower pathway), followed by microautophagic sequestration. However, receptor
function does not distinguish microautophagy and macroautophagy, so Figure 1-1 only
distinguishes non-selective autophagy (upper pathway) from a hypothetical selective
process that might include microautophagy (lower pathway).
Autophagy (macroautophagy) is a subject of active research, producing almost
300 related papers in just the last five years. FDP aldolases are generally abundant,
commonly known, cytosolic enzyme, and the muscle isoform, aldolase A, is
commercially available. Aldolase A and aldolase B have been used as markers for
autophagic uptake of cytosol into lysosomes, and degradation of aldolase by autophagy
is well established (Henell et al., 1987; Kominami et al., 1983; Kopitz et al., 1990;
Seglen and Gordon, 1982; Ueno et al., 1990). Per O. Seglen’s laboratory briefly treated
starved hepatocytes with cycloheximide to prevent new protein synthesis and estimated
degradation rates by loss of enzyme activity. Incubations were short to avoid depletion
of autophagic factors (continual autophagy requires new protein synthesis). A lysosomal
inhibitor (leupeptin) was used to estimate how much degradation occurred in lysosomes.
In this way, starvation-induced degradation of aldolase B was lysosomal occurring at

17
3.6±0.1 %/h. Other cytosolic enzymes with widely different half-lives were similarly
tested. As expected, they had very different total degradation rates. However,
lysosomal degradation (3.3-5.3 %/h) and rates of accumulation in organelles during
lysosomal inhibition (3.1-3.7 %/h) were similar for all the enzymes. These rates match
rates of starvation-induced autophagy (3-5 %/h) and were blocked with 3-methyladenine
a specific inhibitor of autophagy. Thus, Seglen concluded that cytosolic enzymes,
including aldolase B, are degraded via non-specific autophagy (Kopitz et al., 1990).
Interestingly, of all the enzymes tested by Seglen, only two were exclusively degraded in
lysosomes, aldolase B and lactate dehydrogenase H (Kopitz et al., 1990).
Coincidentally, both aldolase B and lactate dehydrogenase H contain sequence motifs for
receptor-mediated targeting to lysosomes for degradation.
Receptor Mediated Targeting to Lysosomes
A pentapeptide sequence (KFERQ) of RNAse A was shown to mediate its
delivery to lysosomes for degradation during nutrient deprivation (Dice and Chiang,
1988). Characterization of this signal identified a motif contained in a subset of cellular
proteins that undergo enhanced degradation in lysosomes during nutrient deprivation
(Wing et al., 1991). The motif has been proposed as a binding site for a molecular
chaperone called HSC73 which then delivers motif-containing proteins into lysosomes in
an ATP-dependent manner (Chiang et al., 1989). The mechanism (Figure 1-1, lower
pathway) also requires intralysosomal HSP70, and a recently identified lysosomal
membrane receptor, LGP96 (Cuervo, et al. 1996). J. Fred Dice proposed that this

18
pathway occurs by a mechanism analogous to transmembrane transport of proteins
during organellar biogenesis (Dice and Chiang, 1988; Terlecky et al., 1992; Wing et al.,
1991).
Recently, Dice’s group made a major advance by identifying a receptor protein in
the target membrane of lysosomes that mediated transmembrane translocation (Cuervo
and Dice, 1996). The lysosomal membrane protein, LGP96, was demonstrated to be a
rate limiting component in this degradative pathway. CHO cells overexpressing human
LGP96 by two to three fold had correspondingly increased degradation of long-lived
proteins. Furthermore, lysosomes isolated from these cells had two to three fold higher
ATP-dependent uptake of the glycolytic enzyme GAPDH (glyceraldehyde 3-phosphate
dehydrogenase), a known substrate for his pathway (Cuervo and Dice, 1996).
Unfortunately, GAPDH does not contain sequence matching previously established
criteria for the receptor-mediated targeting motif. The previous criteria define necessity
for an “essential” glutamine, but in GAPDH, asparagine apparently can substitute for
glutamine (Dice, personal communication). As just described, receptor function has been
demonstrated in living cells; however, evidence for in vivo function of the signal peptide
is lacking.
A recent study found that the conformation of signal motifs were inappropriate to
mediate the receptor-mediated lysosomal targeting pathway (Gorinsky et al., 1996).
Some proteins known to contain motifs for the pathway, including RNAse A, also have
known three dimensional structures. The peptide signal motifs are supposed to be

19
recognized by cytosolic HSC73 which is required for delivery to lysosomes. However,
signals on proteins of known structure are either embedded or occurred in a-helical
conformations. Since hsp70-type chaperones require extended conformations for
recognition, the investigators concluded that HSC73 would required other unknown
factors to relax a substrate protein’s structure and allow signal-mediated targeting to
lysosomes to occur (Gorinsky et al., 1996). The lower pathway in Figure 1-
1 summarizes receptor-mediated targeting to lysosomes, including an unknown factor
that alters the structure of the cytosolic protein.
Except for the work presented in this dissertation, no studies have examined any
aldolase isoform as a substrate for the receptor-mediated pathway. Aldolase A binds
very well to GAPDH presumably for greater glycolytic efficiency (Verlessy and Vas,
1992). Both these proteins appear to be regulated at similar high concentrations in cells
(Verlessy and Vas, 1992), and aldolase B and GAPDH undergo similar starvation-
induced degradation in cultured cells (Kopitz, et al., 1990). Does aldolase B follow
receptor-mediated targeting to lysosomes like GAPDH?
All vertebrate aldolases contain a conserved motif (Fig. 1-2, residues 12-16) for
receptor-mediated targeting of cytosolic proteins into lysosomes (Dice and Chiang,
1988; Zhang et al., 1995). In mammalian aldolase B, two additional sequences for the
lysosomal targeting motif were found (Fig. 1-2, residues 58-62 and 107-111). Whether
any of these three motifs are functional was unknown. Though the lysosomal targeting
motif has rather broad criteria (Dice and Chiang, 1988), an aldolase-sized (40 kD)

20
Q12
ü
MAHRFPALT S EOKKELSEI AQRIVANGKGILAADESVGTMGNR
MAHRFPALTOEOKKELSEI AQRIVANGKGILAADESVGTMGNR
1 Q58
i
LQRIKVENTEENRROFRELLFSVDNSISOSIGGVILFHETLYOKDS
LORIKVENTEENRROFREI LFSVDNSISOSIGGVILFHETLYOKDS
44
^ Qlll
A
OGKLFRNILKEKGIVVGIKLDOGGAPLAGTNKETTIOGLDGLSER
OGKLF RNILKEKGIV V GIKLDOGGAPL AGTNKETTIOGLDGLSER
90
CAQYKKDGVDFGKWRAVLRIS DQCPSSLAIQENANALARYASIC
CAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASIC
QQNGLVPIVEPEVLPDGDHDLEHCQYV SEKVLAAVYKALNDHH
QQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDHH
179
VYLEGTLLKPNMLTAGHACTKKYTPEQVAMATVTALHRTVPAA
VYLEGTLLK.PNMVTAGHACTKKYTPEQV AMATVT ALHRTVPAA
221
VP SICFLSGGMSEEDATLNLNAIYRCPLPRPWKLSFSYGR ALQAS
VPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGKALQAS
265
ALAAWGGKAANKKATQEAFMKRAV ANCQGQYVHTGSSGAAS
ALAAWGGKAANKEATQEAFMKRAMANCKGQYVHTGSSGAAS
300
TQSLFTA SYTY
TQSLFTACYTY
354 364
Figure 1-2: Amino Acid Sequences of Aldolase B Isoforms Used in This Study. The
upper and lower sequences are for rat and human liver aldolase, respectively. Boldface
indicates non-identical residues. Underline indicates pentapeptide signal motifs for
receptor-mediated lysosomal targeting. Large arrows point to essential glutamines of the
signals as indicated (sequence data from Tsutsumi et al., 1984 and Paolella et al., 1984).

21
protein would have only a 7% chance of randomly containing three such signal motifs.
In addition to the motifs, aldolase B has properties similar to other substrates of this
signal-mediated degradative mechanism: (1) long-lived, (2) cytosolic, (3) housekeeping
protein, and (4) degraded in lysosomes by enhanced proteolysis during nutrient
withdrawal. Furthermore, aldolase is closely associated with another glycolytic enzyme
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is an established substrate
for receptor-mediated targeting to lysosomes (Aniento et al., 1993). Aldolase and
GAPDH form a complex that facilitates their sequential roles in glycolysis (Verlessy and
Vas, 1992), both are very abundant, and their in vivo turnover rates are very similar
(Kuehl and Sumsion, 1970), suggesting that they could share degradative mechanisms.
Furthermore, the receptor-mediated pathway proceeds by transmembrane translocation
into lysosomes by a mechanism like that of organellar biogenesis. Coincidentally, the
aldolase isoform of Trypanosoma brucei (45% identical to aldolase B) undergoes
transmembrane transport during biogenesis of the unique glycolytic organelle
of this protozoan (Marchand et al., 1988). Together, these facts suggested that aldolase
B was a likely candidate for receptor-mediated targeting to lysosomes via the proposed
transmembrane transport mechanism.
Ubiquitination and the Degradation of Long-lived Proteins
Ubiquitination is an orderly process whereby a 76-amino acid polypeptide,
ubiquitin, is covalently conjugated to other proteins at its carboxyl terminus. In a series
of transfers, three enzymes (El, E2, and E3) covalently bind and pass ubiquitin to the

22
next protein. El, called ubiquitin-activating enzyme, first conjugates ubiquitin’s carboxyl
terminus. This step is obligatory, and cell lines with temperature-sensitive ubiquitination
have defects traced to mutations in El (Kulka, et al., 1988; Chowdary, et al., 1994). El
transfers ubiquitin to an E2 which transfers it to an E3 which finally conjugates the
ubiquitin to a target protein (sometimes, the E3 step is skipped). Most protein
ubiquitination requires a single El protein, but E2 and E3 enzymes occur as families that
regulate and confer specificity for ubiquitination. This arrangement explains why genetic
defects in general ubiquitination only occur in El enzymes (Ciechanover and Schwartz,
1994; Hochstrasser, 1992).
Cells die without ubiquitination. The process has been implicated in a wide
variety of cell functions reviewed elsewhere (Hochstrasser, 1996; Jentsch, 1992).
Ubiquitination was originally discovered in the rapid degradation of short-lived and
abnormal proteins by cytosolic proteases, and this role remains the best characterized
(Hershko and Ciechanover, 1992). Heat stress causes enhanced degradation of long-
lived proteins in E36 Chinese hamster lung cells. This heat-stress induced degradation
occurs in lysosomes via autophagy and requires ubiquitin-activating enzyme El (Gropper
et al., 1991; Handley-Gearhart et al., 1994). However, specific long-lived proteins that
utilize this ubiquitin-mediated autophagic mechanism have not been identified.
Whether for short-lived or long-lived proteins, ubiquitin-mediated turnover
involves attachment of multiple ubiquitins on a protein targeted for degradation
(Hershko and Ciechanover, 1992). A ubiquitinated protein (a.k.a. ubiquitin conjugate) is

23
a substrate for further ubiquitination, and additional ubiquitins preferentially conjugate to
already attached ubiquitin. A chain of ubiquitins is built on a protein to be targeted. The
multiubiquitin chain then acts as a signal for degradation of the targeted protein. Each
ubiquitin adds an additional 76 amino acids to the protein, and successive intermediates
of multiubiquitination can be demonstrated as a ladder of bands on SDS-PAGE that
contain both ubiquitin and the targeted protein (Chau et al., 1989; Hershko and
Ciechanover, 1992). Multiubiquitination is a well-established signal for stress-induced
degradation of short-lived proteins by a major protease complex in cytosol. Though
total long-lived proteins undergo ubiquitin-mediated stress-induced degradation in
lysosomes, a role for multiubiquitination has not been established for lysosomal
degradation of any specific cytosolic protein.
Recently, work in the laboratory of William A. Dunn, Jr. demonstrated a
connection between ubiquitin and the long-lived protein aldolase B. The evidence
includes data presented in this dissertation, two meeting abstracts, and a manuscript
which has been submitted (Lenk et al., Submitted 1998; Susan and Dunn, 1996; Susan et
al., 1995). Together the data support that aldolase B is multiubiquitinated in vivo and
suggest that ubiquitination is involved with stress-induced autophagic degradation of
aldolase B in lysosomes.
S. E. Lenk and William A. Dunn, Jr. provided the first evidence that aldolase B
has ubiquitinated forms (Figs. 1-3 and 1-4), including a major 68 kD form (Ub68)
enriched in lysosomes during nutrient deprivation (Lenk et al., Submitted 1998; Susan et

Figure 1-3: Characterization of A Major Ubiquitin-Protein Conjugate Enriched in
Autophagic Vacuoles, a) Rats were starved to induce autophagy and lysosomal uptake
of ubiquitinated proteins. Subcellular fractions of liver were prepared, and equal protein
from cytosolic (Cy), lysosome-enriched (Ly), and autophagic vacuole-enriched (AV)
fractions were run on SDS-PAGE, western blotted, and labeled with antibody against a
major ubiquitinated protein (anti-Ub68). Note major bands at 68 kD and cross-reactivity
to a 40 kD protein in cytosol. The 40 kD protein was identified as aldolase B by peptide
sequence analysis, b) Cytosol was circulated on an anti-Ub68 column, washed, eluted,
and preparative SDS-PAGE performed. A gel strip was stained with Coomassie blue R-
250 (CB), and the remaining gel was blotted and cut into strips individually stained with
anti-Ub68 (Ub68) or anti-ubiquitin (Ub). Ub68 and aldolase are indicated at 68 kD and
40 kD, respectively. Arrowheads indicate positions of bands suggestive of intermediates
of multiubiquitination of aldolase (from Lenk, et al., Submitted 1998).

anti-Ub68

Figure 1-4: Amino Acid Starvation Increases Lysosomal Association of Putative
Ubiquitinated Aldolase B via Autophagv. Fao rat hepatoma cells were incubated on
media with or without amino acids (AA) and the autophagy inhibitor 3-methyladenine
(3MA) as indicated. Sub-cellular fractions were collected, and equal protein of
lysosome-enriched fractions were run on SDS-PAGE, western blotted, and stained with
antibodies against Ub68 and ubiquitin (Ub). Positions of molecular weight standards and
Ub68 (arrowhead) are indicated (from Lenk et al., Submitted 1998).

27
205 —
116 —
97 —
â–º
66 —
45 —
29 —
AA
3MA
Anti-Ub68
y üqs
m
#
+
+
Anti-Ub
+
+

28
al., 1995). Aldolase A and B are long-lived proteins known to undergo degradation by
autophagy (Henell et al., 1987; Kominami et al., 1983; Kuehl and Sumsion, 1970; Ueno
and Kominami, 1991; Ueno et al., 1990). It has been determined that amino acid
deprivation (starvation) rapidly enhances autophagy in cultured cells (Kopitz et al., 1990;
Seglen and Gordon, 1982; Ueno et al., 1990). Since Ub68 increases in lysosomes under
similar conditions (Fig. 1-4), the evidence suggested that ubiquitination might play a role
in stress-induced autophagic degradation of aldolase B.
Hypothesis for Stress-Induced Degradation of Aldolase B
The field of protein degradation has made great progress in determining
molecular mechanisms for the degradation of short-lived proteins via a cytosolic protease
complex (the proteasome), however, long-lived proteins which are generally thought to
be degraded in lysosomes have relatively poorly characterized degradative mechanisms.
Cellular degradative mechanisms that respond to environmental changes facilitate
experimental characterization. Starvation (amino acid and serum deprivation) and heat
stress can induce regulated mechanisms for the degradation of long-lived proteins.
Figure 1-1 presents two pathways proposed for the stress-induced delivery of cytosolic
proteins to lysosomes for degradation: autophagy or receptor-mediated targeting (lower
pathway).
In the simplified diagram of Figure 1-1, only topological changes in autophagy
(upper pathway) are shown for a tetrameric cytosolic protein sequestered into an
autophagic vacuole (steps 1 and 2) that fuses with lysosomes (step 3). Heat stress

29
induces autophagic degradation that requires ubiquitination, but specific proteins that are
ubiquitinated during stress-induced autophagy have not been identified. Aldolase B is
known to undergo autophagy, and a putative ubiquitinated form aldolase B associates
with autophagic vacuoles and lysosomes during starvation. To establish a specific
protein for ubiquitin-mediated autophagy, we examined aldolase B as a likely substrate
for ubiquitin-mediated autophagy.
In Figure 1-1, receptor-mediated targeting to lysosomes is also drawn showing
required components (lower pathway). Since established substrates for this pathway
have conformations that would prevent receptor recognition, unknown factors (smallest
circles) have been proposed to relax the structure of substrate proteins (first arrow)
which probably includes disassembly of subunits from quaternary structures (rough-
drawn oval with small circles attached). An exposed signal then mediates assembly of a
complex on the lysosomal surface (second arrow), including the HSC73 chaperone
(medium gray square), the substrate protein (extended coils), the lysosomal membrane
protein LGP96 (darkest rectangle), and possibly other factors (small circles).
Transmembrane translocation (third arrow) also requires an intralysosomal HSP70
chaperone (dark gray square). Aldolase B has characteristics similar to known substrates
for receptor-mediated targeting to lysosomes, but this mechanism was not examined for
any aldolase. Evidence will be shown that ubiquitinated forms of aldolase B have a more
denatured conformation. If aldolase B follows receptor-mediated degradation, then
ubiquitin could represent the unknown factor needed to relax substrate structure.

30
The relationship between receptor-mediated targeting of cytosolic proteins to
lysosomes and ubiquitin-mediated autophagic degradation had not been examined. Since
aldolase B was a potential substrate for both pathways, I hypothesized that during stress,
aldolase B requires both ubiquitination and a receptor-mediated targeting signal for
enhanced degradation in lysosomes.
General Strategy
I adopted the hypothesis that during stress, aldolase B requires both
ubiquitination and a receptor-mediated targeting signal for enhanced degradation. The
two requirements in this hypothesis were separately tested: ubiquitination and a receptor-
mediated targeting signal. In this regard, there were two corresponding aims of this
investigation: Aim #1, perturb ubiquitination and examine the effects on stress-induced
delivery of aldolase B to lysosomes; Aim #2, mutate potential lysosomal targeting signals
and examine effects on starvation-induced degradation of aldolase B.
My first aim was to determine whether stress-induced degradation of aldolase B
requires ubiquitination. Antibodies were raised against aldolase B expressed in and
isolated from E. coli. Since bacteria lack ubiquitin, the antibodies were produced against
antigen that did not contain ubiquitin or ubiquitin-conjugated proteins. With the
antibodies, the presence of aldolase B epitopes in a major 68 kD ubiquitinated protein
(Figs. 1-3 and 1-4, Ub68) and other ubiquitin conjugates was confirmed in subcellular
fractions from rat liver. Epitope-tagged aldolase B was expressed in E36 (parent) and
ts20 (ubiquitination mutant) cells previously used to establish ubiquitin-dependency for

31
heat stress-induced autophagic degradation of long-lived proteins. By examining
changes in the endogenous aldolase A and exogenous aldolase B associated with
pelletable subcellular fractions, evidence was found that these aldolase isoforms require
ubiquitination for autophagic degradation in lysosomes during heat stress. This
supported my hypothesis that stress-induced degradation of aldolase B requires
ubiquitination.
An attempt was made to use protein degradation measurements to confirm that
heat stress-induced degradation of aldolase B requires ubiquitination. Degradation of
aldolase B was found to utilize a temperature-dependent cytosolic proteolytic
mechanism. The cytosolic proteolysis of aldolase B at heat stress temperatures was
similar in magnitude to induced ubiquitin-mediated autophagic degradation. Since the
cytosolic mechanism turned out to be ubiquitin-independent, degradation measurements
could not confirm ubiquitin-mediated degradation of aldolase B via lysosomes.
However, the results demonstrate that mechanisms for degradation of aldolase B include
a novel cytosolic proteolysis.
My second aim was to test whether a receptor-mediated targeting signal was
required for stress-induced degradation of aldolase B A sequence motif has been
defined for targeting cytosolic proteins to lysosomes for degradation during nutrient
deprivation, and aldolase B contains three sequences that match the motif (Fig. 1-2).
Depriving liver-derived cell lines of serum and amino acids causes starvation-induced
degradation of long-lived proteins including aldolase B. Vectors were constructed
expressing epitope-tagged aldolase B and used site-directed mutagenesis to disrupt the

32
putative targeting signals. Wildtype and mutant aldolase B proteins were expressed and
assayed for starvation-induced degradation. Starvation causes enhanced autophagic
degradation of aldolase B expressed in cultured hepatoma cells, and this enhanced
degradation specifically required a targeting signal that includes glutamine residue #111.
This supported my hypothesis that stress-induced degradation of aldolase B utilizes a
receptor-mediated targeting signal.

CHAPTER 2:
MATERIALS AND METHODS
Cell Lines and Culturing
General Maintenance
Except for temperature (see following subsections), all cell lines were maintained
similarly using standard sterile cell culturing techniques. Except where indicated, all
supplies were obtained from Fisher Scientific, Inc. The term “standard culture
conditions” refers to maintenance in DMEM (Sigma #D-5648), 2.2 g/1 NaHCCfy and
10% FBS (Biocell #6201-00) in a 5% CO2 atmosphere, and the standard medium for
stably transfected cells included 0.3 mg/ml active G418 (GIBCO BRL #11811-031).
Cultures were fed every 3-4 days and passaged before complete confluency. For
passages, cell sheets were rinsed with DPBS (Sigma #D-5652) followed by lx trypsin-
EDTA (Sigma #T4174) in DPBS for 4-8 minutes at room temperature or 37°C as
needed. Passages to amplify and maintain cultures for experiments were split 1:10 to
1:50 (area.area), and very fast growing lines that tolerated thin splits were done down to
1 80. Since trypsin/EDTA diluted 1:10 or more with 10% FBS did not affect cell sheets
during 30 min. at 37°C, some thin splits (at least 1:30 into medium with 10%FBS) were
directly plated without pelleting to remove trypsin. For cultures using this short cut,
attachment times, spreading times, growth rates, and experimental results were
unaltered. Passages to replenish frozen stocks were always split heavily at 1:3 to 1:6
from freshly thawed stocks grown to near confluency. For new frozen stocks, cells
33

34
were suspended in medium supplemented with 10% DMSO (Sigma #D-2650), incubated
1-2 h at minus 20°C, then at minus 80°C overnight, and stored at minus 80°C for up to
two months or transferred to liquid nitrogen for longer storage times.
Heat Stress-Inducible E36 Cells and Ubiquitination Mutant
Alan Schwartz kindly provided cell lines: E36 (parent), ts20 (mutant with
temperature sensitive ubiquitin-activating enzyme El), and ts20Elc2 (mutant rescued by
wild-type human El) Chinese hamster lung cell lines. These cells are well characterized
for thermal control of ubiquitin-activating enzyme E1 activity and together have
demonstrated that El-mediated ubiquitination is required for heat stress-induced
degradation of long-lived proteins ((Handley-Gearhart et al., 1994); (Handley-Gearhart
et al., 1994); (Trausch et al., 1993); (Lenk et al., 1992); (Schwartz et al., 1992);
(Gropper et al., 1991); (Kulka et al., 1988)).
Starvation-Inducible Cell Lines
William A. Dunn, Jr. provided Fao (rat hepatoma) and HuH7 (human hepatoma)
cell lines. The Fao cell line originates from a rat hepatocellular carcinoma (Reuber,
1961), and this derivation is well documented (Deschatrette and Weiss, 1974). Fao cells
retain a dozen liver-specific characteristics examined by Mary C. Weiss, including some
endogenous expression of aldolase B (Deschatrette et al., 1979; Deschatrette and Weiss,
1974).
The HuH7 cell line was isolated from a well differentiated carcinoma of a
Japanese man and shown to secrete 16 different plasma proteins associated with liver

35
function (Nakabayashi et al., 1982). Seven expected carbohydrate-metabolizing
activities were present in HuH7 cells, but for two of these, liver-specific isoforms,
pyruvate kinase L and a low-Km hexokinase, were not detected (Nakabayashi et al.,
1982). BHK (baby hamster kidney), and NRK (normal rat kidney) cell lines were
examined briefly during transient transfections.
Plasmid Vector Construction and Mutagenesis
General Molecular Biological Methods
Basic methods were performed essentially as described in Current Protocols in
Molecular Biology (Ausubel, et al., 1994). Except where noted, all supplies came from
Fisher Scientific, Inc. Kits for DNA preparations were from Qiagen and Promega.
Restriction digestions, ligations, other DNA modifications, and PCR utilized supplies
from Promega and New England Biolabs, except as noted below.
PCR Primers and DNA Sequencing
At the University of Florida, the DNA Synthesis Core Laboratory provided all
oligonucleotide primers that William A. Dunn, Jr. or Peter P. Susan designed for PCR.
The University of Florida DNA Sequencing Core Laboratory sequenced parts of
plasmid vectors that were altered, or we did DNA sequencing with a Sequenaseâ„¢ kit
(U.S. Biochemical Corporation).
Expression Vectors for Epitope-Tagged Aldolase B
Kiichi Ishikawa provided pRAB 1710 Amp+ (Tsutsumi et al., 1984), a plasmid
containing the cDNA of rat aldolase B (RAB) used as a PCR template in a reaction
containing two primers (Fig. 2-1). PCR solutions were prepared according to

36
manufacturer’s specifications (Promega) and then run through 40 cycles (each cycle: 1
min. at 94°C, 1 min. at 52°C, and 2 min. at 72°C), yielding DNA coding for rat aldolase
B tagged with the 9E10 myc epitope at the carboxyl terminus (RABM). After
restriction, the product was ligated into Xhol and Xbal sites of the vector pMAMneo-
blue (CLONTECH), yielding pRABM which failed to express RABM. Using EcoKV
and Xbal, I transferred the RABM code to pcDNA3 (Invitrogen), yielding
pcDNA3RABM (Fig. 2-2). William A. Dunn, Jr. provided a pcDNA3-based vector,
pHAHAB which expresses human aldolase B (Sakakibara et al., 1989) with an amino
terminal 12CA5 HA epitope, HAHAB (Lenk et al., Submitted 1998).
a. WID5, 5’ primer: CTCCCTTGGCTCGAGCTGTC
Xhol
Xbal
b. WID9, 3’ primer: TGCTCTAGACTActacaagtcttcttcagaaataagcttttgttcctcGL4GG-
TGTAGGGGCTGTGA
Figure 2-1: Primers for PCR Amplification of Insert Containing cDNA for RABM
Expression, a) 5’primer also called, WID5; b) 3’primer also called WID9. Orientations
are relative to 5’ to 3’ convention. Italics indicate reverse complementary code for
carboxyl terminal amino acids; lower case letters indicate reverse complementary
sequence for myc (9E10) tag; single underline indicates reverse complementary sequence
for a stop codon; boldface indicates Xhol and Xbal restriction sites; other bases are the
same as vector sequences.

37
a.. 5’ DNA Sequence:
-AC1GCKGKA MCAAGCTTA TCGA TA CCGTCGA CCTCGA GCTGTCAATCATG—
EcoR V
Xhol start
methionine
(aldolase B)
b. 3’ DNA Sequence:
myc-epitope code
—ACCTACgaggaacaaaagcttatttctgaagaagacttgTAGTCTAGAGGGCCC—
C-terminal stop Xbal
tyrosine codon
(aldolase B)
C. Carboxyl terminal amino acids of RABM:
—TASYTYEEOKLISEEDL
FIGURE 2-2: DNA Sequence of pcDNA3RABM. a) 5’ insertion site and b) 3’ insertion
site showing new sequence generated by vector construction. Boldface designates
cDNA sequence of rat aldolase B (upper case) and human myc epitope 9E10 (lower
case). Italics designate DNA sequence from the multicloning site of pMAMneo-blueâ„¢.
Standard typeface designates DNA sequence in the multicloning site of pcDNA3â„¢
(Invitrogen). Underline designates indicated restriction sites. Double underline
designates indicated codons. Sequences not shown for rat aldolase B cDNA and
pcDNA3â„¢ vector are available in Tsutsumi, et al. (Tsutsumi et al., 1984) and from
Invitrogen Technical Services, respectively, c) Amino acid sequence predicted for the
carboxyl terminus of the RABM protein expressed from this vector (residues not shown
would be identical to rat aldolase B), single letter amino acid code is underlined for
residues added to create a 9E10 epitope.

Site-Directed Mutagenesis of pcDNA3RABM
38
I used three different strategies for PCR mutagenesis: (1) overlap extension (Ho
et al., 1989), (2) Quick-Changeâ„¢ Site-Directed Mutagenesis Kit (Stratagene #200518),
or (3) restriction-limited insertion as described below (Fig. 2-4; Table 2-1).
I used an overlap extension protocol adapted by Brian Cain from Ho and others
(Ho et al., 1989). In brief, a mutagenic primer pair (see Fig. 2-3, positions 2 & 3, 4 & 5,
or 6 & 7) was made complementary to each other and to base pairs on either side of a
targeted change (non-complementary) in aldolase B coding sequence (Table 2-1). For
example, Q58 mutagenesis started with pcDNA3RABM as template in two PCR
reactions using primers at positions 1 & 2 (Fig. 2-3) to make product for the 5’ end of an
insert and at positions 3 & 8 (Fig. 2-3) to make product for the 3’ end of an insert. PCR
was done for 35 cycles (1 min. at 94°C, 1 min. at 55°C, and 2 min. at 72°C).
At one end of one product, sequence was derived from primer at position 2 and
therefore was reverse complementary to one end of the other product derived from
primer at position 3. These products were combined in Taq polymerase buffer
(Promega), melted at 94°C, and cooled very slowly to allow promiscuous annealing and
yielding a small amount of 3’end-to-end annealed single stranded DNA from 5’ and 3’
products for ends of a desired insert. Taq polymerase was added and the temperature
raised to 72°C for run-off extension through ends complementary to positions 1 & 8
(Fig. 2-3). Primers for positions 1 & 8 were added and cycled through temperatures as
done previously which produced a smear of products due to promiscuous annealing.

39
Rat Aldolase E
c
myc
9E10
\ /
»<■
Xb
FIGURE 2-3: Positions of Primers for Site-Directed Mutagenesis. Map shows the RABM coding region
of pcDNA3RABM; positions drawn to scale. Numbered arrows identify 5’-» 3’ primer sequences at
complementary sites. Shaded areas are cDNA sequences for the indicated polypeptides. Letters B, E,
and Xb indicate restriction sites for enzymes Bsal, EcoR V, and Xba I, respectively. E and Xb are sites
of insertion into pcDNA3 (Invitrogen) multicloning site.
TABLE 2-1: Details of Primers for Site-Directed Mutagenesis
Primer
ID.
Map
Posi¬
tion*
Position (bp)
From
1st Base of
Start Codon
Target
Code
Change
Primer Sequence, 5’to 3’
(sequence non-complementary
to pcDNA3RABM is underlined)
WID17
1
-117 to
-91
+Smal
CTCACT AT AGGGAGACCCGGGCTTGGT
WID19
2
+25 to
+45
Q12(T/N)
CTCCTTCTTA(T/G)TCTCTGAGGT
WID18
3
+25 to
+45
Q12(T/N)
ACCTCAGAGAfA/OTAAGAAGGAG
WID21
4
+163 to
+ 181
Q58(T/N)
CTCGGAAÍT/G1TCCTTCGGTT
WID20
5
+163 to
+181
Q58(T/N)
AACCG AAGGAf A/CVITTCCGAG
WID44
4-
non***
+ 165 to
+195
+SctfI
Q58N
AGAGAAGTACTAAAGAGGAGCTCTCGG
AAATTCCTTCGG
WID43
5-
non***
+196 to
+218
+Pmñ
CGAGACACGTGGACAATTCTATC
WID23
6
+322 to
+339
Q111(T/N)
ACCTCCAfG/TITGTCCAGCTT
WID22
7
+322 to
+339
Q111(T/N)
AAGCTGGACAfA/ClTGGAGGT
WID36
6
+313 to
+340
quit
+Hmd\\\
cacctcctgigtcaagcttgatgcccac
WID35
7
+313 to
+340
+Hindlll
QUIT
GT GGGC ATC AAGCTTG AC AC AGG AGGTG
WID24
8
+617 to
+636
none
AGCAGCCAAGACCTTCTCAG
*Fig. 2-3;**amino acid (single letter code) change by residue # (start M = 1); «‘printer non¬
complementary' to paired primer with position shifted for restriction-limited insertion (Fig. 2-4).

40
Though extraneous products were common, the most abundant product was the desired
mutated DNA fragment representing full length mutated insert. This insert containing
altered DNA code was Bsal digested, gel purified, and ligated into the corresponding
site of fresh pcDNA3RABM from which the wild type fragment was removed.
Normally, restriction sites for primer positions 1 & 8 would be designed for two unique-
site enzymes producing different overhangs. However, Bsal cuts outside its recognition
sequence producing randomly unique overhangs that abrogate a need for separate
enzymes. Bsal cuts a third site near the ampicillin-resistance gene of pcDNA3RABM,
producing two fragments of vector besides the insert fragment. Overhangs for all three
sites were randomly different allowing three-fragment ligation with proper orientations.
The Quick-Changeâ„¢ Site-Directed Mutagenesis Kit (Stratagene) was also used
and found to be more rapid. The manufacturer’s protocols were followed, and primers
designed for primer extension and amplification at Q111 (WID23 and WID22 at
positions 6 & 7) did not work with the Quick-Changeâ„¢ kit. However, longer primers
(WID36 and WID35) were successful. The Quick-Change protocol involves in vitro
synthesis of the entire vector (6.5 kb), possibly introducing errors anywhere in mutated
pcDNARABM. To reduce sequencing, the Bsal fragment containing new mutations was
cassetted into fresh vectors. All altered regions of vectors were sequenced at least twice
to confirm changes in amino acid coding were specific for targeted residues.
For Q58N mutation, a restriction-limited insertion was designed(Fig.2-4). This
method uses a mutagenic primer (WID44) to span the Q58 codon and code for unique

41
blunt-end restriction site, Seal (restriction-limited). Another primer (WID43) was
designed with another unique blunt site, Pmll, such that Seal to Pml I blunt ends ligated
to make proper aldolase B code. Steps were followed as indicated in Figure 2-4 to
produce an expression vector for the mutated RABM, pcDNA3RABMQ58N.
Expressing Epitope-tagged Aldolase B in Cell Lines
Permanent Lines Expressing RABM
E36 and ts20 cells were transiently transfected with pcDNA3RABM, using Lipofectin
(GIBCO BRL) or DOTAP (Boehringer) by the manufacturers’ protocol. When
transfected cultures approached confluency labeled cells occurred in groups of 2 to 8
presumably due to cell division. Transfected cultures were trypsinized at confluency and
diluted >1:15 by area into G418 Medium. Fresh G418 Medium was provided every 1-2
days as needed to remove cell debris and maintain strong selection. By 2-3 weeks post
plating, colonies of resistant cells were isolated, passaged, and screened for RABM
expression (immunofluorescence microscopy and western blotting with anti-9E10
monoclonal antibody as described below).
Different lines permanently expressed RABM at varied levels (~10 fold range on
western blots). Using experimental protocols described below, no effect was seen with
doubling time, degradation of RABM, degradation of total protein, or viability. Of eight
positive clones that continue to express after culture amplification, seven (4 from E36
and 3 from ts20) maintained stable relative levels of RABM for twenty additional
continuous doublings or longer (by immunofluorescence and on western blots, data not

42
target codon
Pmll
WTO 4 3
WID5
, 1
f CAd *
lgtggacaattctatc j
if J
j;
i
I
- GGCI1CCt aAAGGCTCTCGAGGAGAAATCAt„
3' TTA /TgAA^
WTD44 / GaGa
Seal 5'
T
WID9
template
1. PCR with WID5 and W1D44
2. Digest with Xho I and Sea I
I
\
1. PCR with WID44 and WID9
2. Digest with Pml I and Xba I
tcgagct"
CG/
ACC
TGGAGCT
TTTAGT
TCA GTGGACA
CACCTGT
/
CTAGAGG
TCC
TAGT
ATCAGATC
3. Ligate to make pcDNA3RABMQ58N
J
pcDNA3RABM digested with Xho I and Xba I
Figure 2-4; Restriction-Limited Insertion for Constructing pcDNA3RABM058N.
Above, primers (small arrows and sequences for WID43 and WID44) are shown relative
to pcDNASRABM template (bar). Sequences juxtaposed to template indicate
complementary regions. Relative positions of Q58 codon and restriction sites are
indicated. Below, DNA pieces for a three-fragment ligation. See text, Fig. 2-3, and
Table 2-1.

43
shown). To facilitate experimental quantification, the highest RABM-expressing E36
and ts20 cell lines fully designated E36RABM14.1 and ts20RABM10.2 or abbreviated
throughout this dissertation as E36AB and ts20AB, respectively, were used. In control
experiments, variation in the level of RABM expression did not affect results (data not
shown).
Similar procedures were repeated with Fao rat hepatoma cells transfected, except
another plasmid pHAHAB also was used to express human aldolase B tagged with the
12CA5 HA epitope at its amino terminus (HAHAB). Screening for HAHAB expression
was done with monoclonal antibody against 12CA5. When G418-resistant clones were
isolated at very most 20% of cells in a given clone had visible expression that was mostly
dim with a few bright cells, and this fraction was rapidly lost with culture splitting for
amplification. Subcloning and screening of a few hundred colonies produced one truly
stable line expressing HAHAB at levels comparable to ts20AB expression of RABM.
This line was designated FaoAB. FaoAB cells split at low density (1:20 or less) grew
much slower than parent Fao cells. In passages using about 1:10 or 1:15 splits, growth
rates were similar to parent Fao cells. An attempt to isolate HuH7 cell lines expressing
RABM was made, but failed to produce any clones having permanent expression.
Transient Transfection System
Fao, HuH7, NRK, and BHK (hepatic and renal cell lines derived from tissues that
express aldolase B) were transfected for transient RABM expression with a series of
lipids according to manufacturer’s standard protocols (Invitrogen and Boehringer-

44
Mannheim). Transient transfection gave very broad cell-to-cell variation in expression by
9E10 immunofluorescence, but ratios of bright to dim cells were relatively reproducible
between transfections. Transfection efficiency was defined as fraction of labeled cells.
Immunofluorescence
Cells were grown on glass coverslips to desired confluency, rinsed briefly with
PBS and fixed with 4% paraformaldehyde in PBS for 20-30 minutes. Fixed cells were
washed three times for 10 minutes in 50 mM ammonium chloride/ 0.1% Tx-100/PBS.
Coverslips were placed on drops containing antibody diluted 1:100 in 5%NGS/ 0.1%
Tx-100/PBS for 1-2 hours at room temperature. Coverslips were washed four times for
5 minutes in 0.1% Tx-100/PBS. Coverslips were placed on drops containing an
appropriate secondary antibody (rhodamine or fluorescein conjugated) diluted 1:100 in
5%NGS/ 0.1% Tx-100/PBS for 1 hour at room temperature. Coverslips were washed
six times for 5 minutes in 0.1% Tx-100/PBS, then mounted on Fluoromount G (GIBCO
BRL).
Antibodies
Preparation of Ubiquitin-Free Aldolase B Antigen
Different E. coli strains were transformed with pXPB, a plasmid vector kindly
provided by Dean R. Tolan for bacterial expression of enzymatically active human
aldolase B (Beernink and Tolan, 1992). The aldolase B expressed in E. coli retains all
the enzymatic properties of the protein isolated from human liver (Sakakibara et al.,

45
1989). Increased expression of a 40 kD Coomassie signal in SDS-PAGE of whole cell
preparations was apparent in transformed cells.
E. coli JM83 cells were transformed with pXPB, which produced much more 40
kD protein than untransformed cells, about 0.3 mg per ml of 1.9 OD6oo nm culture. A 250
ml culture of LB broth + ampicillin (100pg/ml) was grown to 1.9 OD6oo nm culture and
pelleted in a Beckman GSA rotor at 3500 rpm for 10 min. Samples were maintained at
0-4°C for the rest of the procedure. The cell pellet was resuspended in 20 ml 15%
sucrose/50 mM EDTA/50 mM Tris-HCl pH 8.5. To this, 5 ml 5 mg/ml lysozyme was
added, gently mixed by inverting, and incubated 15 min. Then 15 ml 0.1% Triton X-
100/50 mM Tris-HCl pH 8.5 was added, gently mixed, and incubated with periodic
inverting for 20 min. After centrifugation at 9000 rpm for 30 min. (GSA rotor), the
supernatant was decanted into Polyclear tubes and ultracentrifuged in aSW27 rotor at
23,000 rpm for 60 min. The resulting supernatant was the crude extract which was
further processed as previously described for isolation of aldolase B from liver extracts
(Penhoet and Rutter, 1975). Saturated (NEL^SCL was slowly added (0.5 ml/min.) to
45% final concentration with constant stirring. After centrifugation at 9000 rpm for 60
min., the supernatant was collected and the pellet discarded. (NH^SC^ was slowly
added to 60% concentration, and 6 N NH4OH added to pH 7.5. The mixture was
immediately transferred to centrifuge bottles and let stand for >2 hours. After
centrifugation at 9000 rpm for 60 min., the 60% (NTL^SCL pellet was dissolved in 1
mM EDTA/10 mM Tris-HCl pH 7.5 and dialyzed against the same buffer. The sample

46
was loaded onto a 25 ml phosphocellulose (fine mesh, 1.26 meq/g) column prepared
exactly according to Penhoet and Rutter. The column was washed with 5 mM EDTA/50
mM Tris-HCl pH 7.5 (50-60 ml) until OD280nm approached zero. Then 2.5 mM fructose
1,6- diphosphate in 1 mM EDTA/10 mM Tris-HCl pH 7.5 was used to specifically eluted
a sharp aldolase peak. Peak fractions with specific activities of 0.83 to 0.98 aldolase
U/mg (1 U/mg expected for aldolase B) were pooled, precipitated by 55% (NFL^SCL,
and stored as a suspension at 4°C. Before immunization, the suspension was dialyzed
into 10 mM Tris-HCl pH 7.5. According to Coomassie labeled SDS-PAGE and
enzymatic properties (Rutter et al., 1966), the recombinant human aldolase B constituted
at least 95% of the final protein and was more than 99.99% pure of bacterial aldolase
activities, containing <0.005% bacterial isoform (EDTA-sensitive) activity.
Production of Antibodies Against Aldolase B
Rabbits were fed and housed by University of Florida Laboratory Animal
Services. Antibodies to native and denatured aldolase B were raised as previously
described (Reznick et al., 1985) except ubiquitin-ffee human aldolase B antigen was used
(as prepared above). For making antibody to native aldolase B, 50 pg antigen in 0.5 ml
10 mM Tris-HCl pH 7.5 was emulsified 1:2 with complete and incomplete Freund’s
adjuvants for immunizations and boosts, respectively. For antibody to denature antigen,
the antigen solution was supplemented with 2%SDS/2%PME and boiled 10 min. prior to
mixing with adjuvant and immediately before administration to animals. Intradermal

47
injections were in the thoracic region on the backs of specific pathogen-free New
Zealand White rabbits. The first boost was two weeks after initial immunization.
Preparative Western blots of rat liver cytosol were routinely used to follow
specific immunoreactivities. One week after the first boost, the rabbit receiving native
antigen produced a highly reactive serum specific for 40 kD aldolase B which was
maintained without further boosts. Unless otherwise specified, these antibodies were
used to detect native aldolase B-specific polyclonal epitopes throughout this study.
One week after the first boost, the rabbit receiving denatured antigen produced
antibody that specifically labeled a 68 kD protein moderately, 60 kD and 78 kD proteins
lightly, and a high molecular weight smear. This pattern was remarkably similar to that
for anti-Ub68. Furthermore, relative recognition of isoforms A and B were comparable
to that for Ub68. Thus, denaturation of aldolase B disrupted isoform-specific epitopes
and produced anti-denatured aldolase B that similarly recognized epitopes in both
aldolases A and B. Interestingly, the early bleeds had little or no reactivity to 40 kD
aldolase.
Every 4-7 weeks (after injection sites completely healed), the rabbit immunized
with denatured aldolase B was boosted. After the second boost, antibody
immunoreactivities were greatly increased. Though these sera recognized some non¬
protein epitopes, they retained specificity for proteins labeled with sera from earlier
bleeds. After the third boost, sera recognized 40 kD aldolase B but never as well as
antibody to native antigen.

48
Other Antibodies
Anti-Ub68 was provided by William A. Dunn, Jr. and is a polyclonal rabbit
antibody raised against a major ubiquitin-protein conjugate purified from lysosomes
(Lenk et al., Submitted 1998). Monoclonal mouse antibodies against thel2CA5 HA and
9E10 myc epitopes were obtained from the University of Florida Hybridoma Core
Laboratory. Alternatively, hybridoma cells expressing anti-9E10 (provided by the
Hybridoma Core) were injected into the peritoneum of BALB/c mice (provided by the
University of Florida Laboratory Animal Services Division), and periodically, ascites was
harvested until mice showed signs of discomfort or disease at which time they were
euthanized.
Viability Assays
All treatments were measured for viability except for incubations in HBSS
(Hank’s Balanced Salts Solution) which were used to induce cell death. Heat stressed
cultures using HBSS had many cells rounding and sloughing off culture surfaces in 4 to 6
hours of treatment, and were only used in experiments to determine the effect of dying
cells on protein degradation measurements. When HBSS was replaced with MEM
(Sigma #M-0268 + 2.2 g/1 sodium bicarbonate), such rounding and sloughing was
delayed beyond 25 hours of treatment. To measure metabolic viability, cultures exposed
to each experimental treatment were recovered under normal maintenance conditions for
12 hours followed by addition of the same labeling medium (0.1 mCi j5S-methionine/ml)
used for protein degradation assays. Then protein synthesis was measured as TCA

49
precipitable counts incorporated in 20 min. Metabolic viability was defined as the %
protein synthesis relative to duplicate cultures treated with fresh medium under normal
maintenance conditions. Since experimental treatments (see Experimental Conditions)
lack serum and nutrients (MEM instead of DMEM) relative to maintenance conditions,
our assay probably underestimates metabolic viability. In MEM, more than 80% of
metabolic viability was retained for 28 hours and 15 hours in heat stressed E36 and ts20
cells, respectively. Unless otherwise stated, all data are reported for incubations and
drug treatments that retained 90% or greater metabolic viability. For low density
cultures (<10% confluent), the Cell Titer 96 AQ System" was used according to
manufacturer’s protocols (Promega) which indirectly measures electron transport
pathway activity.
Subcellular Fractionation
Subcellular fractionation of E36 and ts20 cells is summarized in Figure 2-6. To
produce a homogenate containing intact organelles, cells were grown to recent
confluency in 100 mm dishes, using 10 ml DMEM (Sigma #D-5648) + 10% FBS
medium. Culture rinsed with 10 ml DPBS (Sigma D-5652) was treated with 1.0 ml IX
Trypsin-EDTA (Sigma #T-9395 diluted in DPBS),and incubated at room temperature
until cells easily and completely knocked loose from the plate (10 minute maximum).
After adding, 5 to 10 ml medium containing 10% FBS, cells were transferred to 15 ml
conical centrifuge tubes (polypropylene), centrifuged at 1,500 X g for 5 minutes, and
supernatant discarded. Pellet was suspended in 1.0 ml ice-cold cavitation buffer (SHE):

Figure 2-6: Subcellular Fractionation Scheme for E36 and ts20 Cells
Cell Culture
4
Scrape cell sheet
4
Centrifuge 1000 x g v 0
—r Supernatant = !SC
I
Pellet
4
Homogenize _v T T
r Homogenate = HO
Centrifuge 1000 x g
4
Supernatant
4
—> Pellet
4
Pool = LP
t
Centrifuge 1000 xg peUet
4
Supernatant = LS
4
Centrifuge 100,000 x g —^ Supernatant = HS
4
Pellet
4
Resuspend
4
Centrifuge 100,000 x g
4
Pellet = HP
Supernatant (wash , dilute HS) —> <1% of culture content
• 50

51
250 mM sucrose, 10 mM HEPES (Research Organics #6003H-3), 1 mM EDTA, pH 7.4,
loaded into a N2-cavitation bomb chamber using 2-3 ml total SHE volume, pressurized
to 65 psi for 10 minutes, and collected sample from bomb directly into a pre-chilled
Dounce homogenizer. Procedural details were as described by the cavitation bomb
manufacturer’s specifications (Kontes). The sample was homogenized with 5 strokes of
a pestle, minimizing froth by limiting passage of bubbles to the sample side of pestle.
This was saved as the homogenate (Ho).
Alternatively, the trypsinization step was replaced by scraping the cell sheet
directly into SHE which resulted in a large fraction of cytosol but not organelles to leak
out. This facilitated the separation of aldolase associated with organelles from soluble
aldolase in the cytosol. Scraped cells were pelleted as done above to remove trypsin
solution, but in this case the supernatant was saved (to assess cytosolic leakage) as the
scrape fraction (Sc). The rest of cavitation and homogenization was performed as
above.
Homogenate (Ho) was fractionated essentially as described previously
(Rickwood, 1992; Coligan et al., 1995), using Sigma reagents for assays and Sorvall or
Beckmann centrifuges and accessories. Homogenate was centrifuged at 1,500 X g for
20 minutes yielding a low speed pellet (LP) and supernatant. The centrifugation was
repeated with the supernatant to make sure nuclei, large debris, and unbroken cells were
efficiently removed, the resulting pellet was pooled in LP, and the resulting low speed
supernatant (LS) was centrifuged at 100,000 X g for 90 minutes, yielding a high speed

52
supernatant (HS) and pellet. The pellet was resuspended in well over 400 volumes of
fresh SHE and centrifuged at 100,000 X g for 60 minutes. The supernatant had a
content similar to HS but was much more dilute (data not shown), so it was not pooled
with HS. The pellet was saved as the high speed pellet (HP).
Fractionation conditions were developed to separate abundant cytosolic aldolase
from that associated with organelles. An initial scrape into fractionation buffer caused a
three fold greater leakage of aldolase than acid phosphatase, so this step was retained in
the procedure. Conditions were chosen to maximize recovery of lysosomal organelles
(acid phosphatase and (3-hexosaminidase) from LP to HP and minimize release of
lysosomal markers into Sc and HS. Recovery was reasonable (85-99% accounted).
Enzyme Assays
Aldolase
Aldolase assays were performed as previously described (Penhoet and Rutter,
1975). “Aldolase reaction mix” includes 50 pi 6.3 mg/ml a-glycerophosphate
dehydrogenase-triose phosphate isomerase mixture + 4 mg NADH (99% pure, Sigma) +
20 ml 0.1 M glycylglycine pH 7.5. Add 5-50 pi of sample to 1 ml of aldolase reaction
mix, and measure background AOD (340 nm)/min. (BG); add 50 pi 50 mM fructose-1,6-
diphosphate (FDP) or lOOpl 100 mM fructose-1-phosphate (F-l-P), and measure assay
AOD (340 nm)/min. (ASSAY). Aldolase activity in units, U = (ASSAY-BG)/12.44 for
FDP or = (ASSAY-BG)/6.22 for F-l-P.

53
Acid Phosphatase
Acid Phosphatase activity was the OD (405 nm) in lOOOx g supernatant after 60
minute incubation at 37°C for 50 pi sample in 200 pi 8 mM p-nitrophenolphosphate/
2mM MgCl2/ 90 mM Na-acetate pH 5.0 stopped by 600 pi 0.25 M NaOH (Rickwood,
1992).
Protein Analysis
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel
staining and drying, western blotting, and autoradiography followed standard protocols
similar to those described and referenced elsewhere (Ausubel et al., 1994; Coligan et al.,
1995). Gels were made with 10% polyacrylamide (1:35 bis-acrylamide).
Immunochemiluminescent detection was done with Amersham ECL Reagents and
protocols, except blots were rinsed in Kodak 1XCDS buffer just before exposure to ECL
chemicals. Protein concentrations were determined with Bio-Rad protein assay (IgG
standard) reagent or spectrophotometric absorbence (205 nm or 280 nm).
For direct immunoprecipitations, monoclonal antibody was purified and
conjugated to sepharose 4b by a CNBr protocol similar to that described in Coligan et
al., 1995. Cells were incubated on a shaker at 0-4°C with ice cold minimum lysis buffer
(MLB: 1%NP40/ 1 mM EDTA/50mM Tris-HCl pH 7.4), standard lysis buffer (SLB:
150 mM NaCl/ MLB), or modified radioimmunoprecipitation assay buffer (mRIPA:
0.1% SDS/ 0.25% desoxycholate/ SLB) supplemented with a cocktail of protease
inhibitors (leupeptin, TLCK, pepstatin, aprotinin, and PMSF obtained from Sigma or

54
Boehringer/Mannheim) at concentrations according to Harlowe. The mRIPA gave the
best results with mAb’s conjugated to sepharose, and SLB gave the best results with
polyclonal sera precipitated with commercial protein A-agarose (Boehringer-Mannheim).
Lysates were precleared for 15-20 minutes on maximum in a microfuge then transferred
to tubes containing 4-10 pi packed bed volume of mAb-sepharose or 1-5 pi antiserum
then rotated in the cold. After one hour, tubes containing antiserum only received 10 pi
packed bed of protein A-agarose. Rotating incubation was continued for varying times,
usually overnight, and such times were held constant within a given experiment. Rapid
washes by quick microcentrifugation and aspiration of supernatants were done three
times with the same lysis buffer and one time with TBS, then immediately processed for
other procedures. For SDS-PAGE, a 2-fold concentration of sample buffer was directly
applied to the pellet and heated at 95-100°C for 5-10 minutes. For precipitable aldolase
activity, pellets were further washed with TE pH 7.5 then resuspended in aldolase
reaction buffer.
Densitometry (autoradiographs, chemiluminographs, & Coomassie gels) were
quantified using a desktop scanner and Sigmagel software. Automatic brightness and
contrast settings determined initial settings that were then kept constant. Spot settings
were chosen to encompass regions of interest, reduce background, and optimize signal.
Standard curves were routinely performed to characterize linear response ranges for
relative protein levels which also defined backgrounds and allowed quantification of
relative signals. Where protein bands were specific for transfected cell lines,

55
untransfected cells were run in parallel through procedures and quantified to establish
backgrounds.
Stress-Induction of Protein Degradation
Culture Preparation
Cells were plated, grown, and maintained at confluency for 2-3 days. About 30-
40 hours before an experiment, cultures were fed with standard maintenance medium or
for protein degradation assays, 14C-Valine or 35S-Methionine was made up in comparable
medium with the corresponding unlabeled amino acid omitted. At the beginning of an
experiment, cells were switched to the media and temperatures indicated in Table 2-2.
Table 2-2: Comparison of Systems for Stress-Induced Degradation of Proteins
Heat Stress Induction
Starvation Induction
Control
Stress
Control
Stress
Medium
MEM
MEM
DMEM+FBS
KH*
Temperature
30.5°C
39.5°C
37°C
37°C
*KH, Krebs-Heinseleit medium (Lefer et al., 1982)
Heat Stress
Cultures were prepared as described above and replicates were incubated under
control temperature (CT) and heat stressed (HS) conditions (Table 2-3) which are
permissive and non-permissive, respectively, for the ubiquitin-activating enzyme El of
ts20 cells (Handley-Gearhart et al., 1994; Kulka et al., 1988; Lenk et al., 1992).
Accordingly, CT conditions included 4 mM bicarbonate-buffered MEM under 5% CO2 at
30.5°C, and HS conditions included 20 mM HEPES-buffered MEM under air at 41.5°C
for 1 hour followed by 39.5°C. These incubations followed established protocols for the

56
E36/ts20 cell system, except MEM replaced HBSS to improve viability. In some
experiments, media were supplemented with 5-10 mM 3-methyladenine (3MA) or 40-
160 pM chloroquine (CHQ) as indicated in Results. Logistics and consistency with
published protocols required differences in buffering and atmosphere between CT and
HS conditions. As a control for such
Table 2-3: Heat Stress (HS) and Control Temperature (CT) Treatments
to Determine Ubiquitin-Activating El Mediated Processes
Experimental
Condition
MEM buffer*
Temperature
Incubator
CT
2.2 g /I NaHCOj
30.5°C
standard
(Permissive)
pH 7.4
5% C02
HS
20 mM HEPES
1 hour @ 41.5°C
submerged
(Non-permissive)
pH 7.4
then 39.5°C
in water bath
Repeat treatments with two cell lines: E36 (parent) and ts20 (mutant)
*For protein degradation experiments, medium was supplemented with unlabeled amino
acid corresponding to that used for labeling (5 mM methionine or 10 mM valine),
differences, CT as summarized in Table 2-3 was compared with CT in HEPES-buffered
MEM under air, yielding no differences in cell morphology, viability, or protein
degradation measurements (data not shown). The other control (i.e. comparing HS in
conditions above with HS in bicarbonate-buffered MEM under 5% CO2) was not tested,
because bicarbonate buffering varies with temperature.
Starvation (Nutrient Stress)
Cultures were prepared as described above and replicates were refed with fresh
standard maintenance medium (DMEM + 10 % FBS) or Krebs-Heinseleit (KH) medium

57
and referred to as “Fed” or “Starved,” respectively. KH components are given in Lefer,
et al., 1982. For protein degradation experiments, media were supplemented with
unlabeled amino acid corresponding to that used for labeling (5 mM methionine or 10
mM valine). Inhibitor treatments utilized the same levels as for heat stress above.
Protein Degradation
Permanent cell lines or transiently transfected cultures were treated according to
instructions in the section Stress-Induction of Protein Degradation to produce cells with
metabolically labeled proteins containing '5S-methionine or 14C-valine. Cell sheets were
routinely rinsed with DPBS (Sigma) immediately followed by application of media
containing unlabeled excess amino acid (5 mM methionine or 10 mM valine) to cultures
described above. This initiated chase of radiolabel incorporated into proteins.
At various times, aliquots of media were collected and TCA precipitated to
measure release of soluble counts measured with a scintillation counter. At the end of
the chase, whole cultures were TCA precipitated to determine total counts. Fraction of
TCA soluble counts released at various times were subtracted from 1 to calculate TCA
precipitable counts, representing the remaining total radiolabeled protein at those times.
Alternatively, cultures were harvested at each time point, processed for
immunoprecipitation, SDS-PAGE, and autoradiography, and the radioactive signals in
specific protein bands (absent in untransfected cells) were quantified by densitometry,
representing the remaining radiolabeled protein (aldolase) at those times.

58
To compare degradative rates for proteins expressed at different concentrations,
relative rates are normalized to the initial amount of the protein, giving a fractional
change per time with units %/hour. This fractional rate is constant for unchanging
degradative mechanisms regardless of a substrate protein’s concentration. For a given
protein, this fractional rate defines the degradative rate constant, kd. For total proteins,
the fractional rate represents weighted average kd contributed by the varied amounts of
different proteins. Throughout this dissertation, all degradative rates and other rates of
protein decrease are estimated with the following calculation. Fractions of radiolabeled
protein remaining were transformed by the natural log (In) and regression analysis
performed using the following function:
ln(100*St/S0) = -kd • t,
where St = signal at time t, S0 = initial signal, kd = first order degradative rate constant,
and t = time. Degradation rates were taken as the negative slope of the regression (kd)
and the standard error of the slope was calculated as the standard error of y at x divided
by the square root of the deviations squared of x. Degradative turnover is also described
by half-life, ty„ the time needed to replace 50% of existing molecules with new ones. In
general, the degradative rate constant and half-life are simply converted by kd = ln(2)/ ty2
= 0.693/1>/2. Note that some investigators do not follow the empirically confirmed first
order relationship of kd and ty2. This results in kd and ty2 reported in the literature that can
vary by as much as three fold from similar data presented using conventional
calculations.

CHAPTER 3:
UBIQUITINATION MEDIATES LYSOSOMAL PROTEOLYSIS OF ALDOLASE B
Introduction
Aldolase B undergoes degradation during stress via autophagy. In Chapter 1,
evidence was described for ubiquitin protein conjugates which underwent starvation-
induced enrichment in autophagic vacuoles and lysosomes, and preliminary work
suggested that putative ubiquitinated aldolase B was amongst these conjugates (Figs. 1-3
and 1 -4). This resulted in the hypothesis that stress-induced autophagic degradation of
aldolase B requires ubiquitination. In the first part of this chapter, the presence of
aldolase B in ubiquitin conjugates is confirmed, and a role for ubiquitination in the heat
stress-induced autophagic degradation of aldolase B is demonstrated.
Two mechanisms were found to simultaneously mediate enhanced degradation of
aldolase B during heat stress: autophagy and cytosolic proteolysis. To separately
examine effects on autophagy, heat stress-induced changes in endogenous aldolase A and
exogenous epitope-tagged aldolase B associated with pelletable organelles were assayed.
In addition, autophagic degradation of long-lived proteins was demonstrated to require
ubiquitination. The results described below support a role for ubiquitination in the
function of a subset of lysosomal proteases.
59

In Vivo Multiubiquitination of Aldolase B
60
Previously, antibodies were raised to a major ubiquitin-conjugated protein, Ub68,
that associated with autophagic vacuoles and lysosomes during stress-induced
autophagy. On western blots of subcellular fractions isolated from rat liver, anti-Ub68
produced a pattern suggestive of a 40 kD protein successively conjugated with serially
increasing numbers of ubiquitin. Such a pattern is referred to as a ubiquitin ladder for
the modified core protein. Peptide sequence analysis identified the 40 kD protein as
aldolase B, suggesting that anti-Ub68 reactive proteins might represent a ubiquitin ladder
for aldolase B.
To confirm the existence of ubiquitinated forms of aldolase B, antibodies (Fig. 3-
2) were raised against aldolase B (Fig. 3-1) and used to assay western blots of rat liver
fractions from the previous studies (Fig. 3-3). In short, anti-aldolase B recognized the
same proteins as anti-Ub68, confirming a ubiquitin ladder for aldolase B and
demonstrating that aldolase B is multiubiquitinated in vivo. The data are consistent with
ubiquitin-mediated autophagic degradation of aldolase B.
First, antibodies against aldolase B had to be produced. However, aldolase B
isolated from animal cells would need to be purified away from contaminating ubiquitin
and ubiquitin conjugates. Since bacteria lack ubiquitin, aldolase B was expressed in E.
coli and purified to produce ubiquitin-free antigen for immunization (Fig. 3-1). E. coli
strains transformed with an expression vector for human aldolase B (Fig. 3-la, lanes x)
produced more 40 kD protein (expected size of aldolase B) relative to untransformed
cells (Fig. 3-la,

Figure 3-1: Isolation of Ubiquitin-Free Aldolase B Expressed in E. coli. a) Three E. coli
strains DH5a, JM109, and JM83 were transformed for aldolase B expression as
described in the text, pelleted, suspended in sample buffer, boiled, and run on SDS-
PAGE with 60 pi culture (OD60o nm = 1.5) equivalents per lane, u and x designate
untransformed and transformed cells, respectively. The 40 kD band specific for
transformed cells is indicated; b) E. coli JM83 cells expressing aldolase B were
fractionated as described in the text: 1 and 2, whole cell preparations (as in part a)
untransfected and transfected, respectively; 3, crude extract; 4, 45% (NFLO2SO4
supernatant; 5, 45-60% (NFL^SCL cut; 6, aldolase activity peak from phosphocellulose
column; 7, dialyzed antigen ready for immunization; 8, rat liver cytosol used to screen
antibodies; 9, detection of lane 8 with antibody raised against protein in lane 7. Lanes 3
to 6 were loaded with 12 pg, lane 7 with 4 pg, lane 8 with 25 pg, and lane 9 with 10 pg
of protein. Dark bands on light background indicate Coomassie R-250 label in gels, and
light bands on dark indicate western blotted proteins detected with anti-native aldolase B
by ECL (Amersham); c) Elution profile for phosophocellulose chromatography, Relative
Amounts: Protein, OD(280 nm); Aldolase Activity, mU/10 (loaded then started washes
when collecting fraction 9); FDP i, elution started with fructose 1,6-diphosphate.

Relative Measurement
DH5cx JM109 JM83
62
¿I. U X U X u
¿4ܧ§r Sr:
40â–º^.r
S p #1 «i ■
c.
Fraction Number

Figure 3-2: Antibodies Against Aldolase B. a) A preparative western blot of rat liver
cytosol run on SDS-PAGE (see Coomassie in Fig. 3-6b, lane 8) was prepared and strips
containing approximately 3 pg total protein were probed with sera from rabbits
immunized with native and denatured aldolase B as indicated, each lane contains a strip,
numbers above each lane correspond to bleed numbers, and numbers with arrowheads
indicate molecular weight, b) A Bio-Rad slot-blotting apparatus was used to load 10
ng/slot of antigen indicated by vertical labels (Aldolase A from Sigma), each row was
probed with serum raised against antigen indicated by horizontal labels with increasing
serum dilutions shown at the bottom of the figure, corresponding preimmune sera were
used in rows immediately above and below anti-aldolase B and anti-Ub68, respectively.

Native
Denatured
64
0 1 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13
â—„ 68
â—„ 40
b
Serum
50 200 800 3200 12800

Figure 3-3: Aldolase B Ubiquitinated In Vivo is Enriched in Lysosomes. a) Preparative
SDS-PAGE of cytosol from starved rat liver was western blotted onto nitrocellulose
then cut into strips with ~3 pg protein/strip, and stained with early antisera against
aldolase B (Fig.3-2a, bleed 4) or Ub68 (bleed 5); b) Aldolase B antisera from bleeds
after booster injections (Fig.3-6a, bleed 10) were reacted with Cy (cytosol strips as
prepared above) or ML (similar strips using a lysosome-enriched fraction instead). N,
antiserum to active native enzyme; N0, same as N with 10-fold longer ECL exposure; D,
antiserum to chemically denatured enzyme; I, antiserum to antigen extracted from
polyacrylamide gel slices; and P, preimmune serum. Arrowheads, molecular weights in
kD. Dots on rightmost edge indicate bands at molecular weights higher than expected
for 40 kD aldolase B subunit. Susan E. Lenk provided subcellular fractions defined as
follows: a 1,000 x g supernatant of rat liver homogenate was centrifuged at 6,000 x g;
the resulting pellet was enriched in lysosomes and contained mitochondria (ML); the
6,000 x g supernatant was centrifuged at 100,000 x g yielding a supernatant fraction
referred to as cytosol (Cy).

Cytosol
Aldolase B
Aldolase B Ub68
a. N D P I P
1 N N N0 N0 D D
D. CyML Cy ML Cy ML

67
lanes u). Aldolase B was purified from the most productive strain, JM83, by cellulose
phosphate chromatography (Fig. 3-lc) of a 45-60% ammonium sulfate cut (Fig. 3-lb,
lane 5) from crude lysate (Fig. 3-lb, lane 3). Cellulose phosphate chromatography
separates aldolase B by substrate affinity at the enzyme’s active site, allowing
enzymatically active aldolase B to be specifically eluted with fructose 1,6-diphosphate,
FDP (Fig. 3-lc, peak at fraction #30). Aldolase B in peak fractions was at least 95%
pure based on Coomassie stained SDS-PAGE gels (Figure 3-lb, lane 6) and specific
activities ranging 0.95-0.98 U/mg (pure aldolase B = 1.0 U/mg). EDTA resistance of
purified aldolase B activity indicated that contamination by class II bacterial aldolase was
less than 0.005% (data not shown).
Aldolase B antigen described above was used to raise antibodies against native
and chemically denatured aldolase B as detailed in Materials and Methods (Chapter 2).
Preparative Western blots of rat liver cytosol were routinely used to follow specific
immunoreactivities (Fig. 3-2a). Native antigen produced a highly reactive serum specific
for 40 kD aldolase B. Anti-native aldolase B demonstrated minimal cross-reactivity
with aldolase A (Fig. 3-2b). However, this antibody aldolase B effectively recognized
aldolase B from different animal species(Fig. 3-2, a. 40 kD rat aldolase B, b. purified
human aldolase B).
A rabbit immunized with denatured aldolase B produced antibodies (Fig. 3-2a
“Denatured” bleeds 2 through 5) that specifically labeled a pattern indistinguishable from
that for anti-Ub68 on western blots of subcellular fractions from rat liver (compare Fig.

68
3-3, lanes D with Figs 1-3 and 1-4, anti-Ub68). This demonstrated aldolase B epitopes
in previously identified ubiquitin-protein conjugates and confirmed that aldolase B is
ubiquitinated in vivo. The antibody to native aldolase B was specific for the 40 kD
unmodified monomer. However, with ten-fold greater exposure times, even the anti¬
native aldolase B detected a ubiquitin ladder (Fig. 3-3b, N„). Preimmune sera failed to
label any proteins. Antibodies raised against native aldolase B are known to be highly
specific (Haimoto et al., 1989). Given this, labeling with anti-native aldolase B provides
even stronger evidence that aldolase B is multiubiquitinated in vivo, and confirms that
Ub68 is probably a stable conjugate of the form: (aldolase B)i(Ub)4.
Multiubiquitinated Aldolase B is Denatured and Enriched in Lysosomes
Aldolase A and B can spontaneously refold to active enzyme after reversible
denaturation treatments (Horecker, et al., 1972; Beemink and Tolan, 1996). Anti¬
denatured aldolase B preferentially recognized ubiquitinated (> 40 kD) forms, whereas
anti-native aldolase B preferentially recognized the unmodified (= 40 kD) form (Figs. 3-
2a and 3-3). These results indicated that ubiquitination inhibits spontaneous refolding of
aldolase B into native conformations. Reznick and Gershon also raised antibodies
against native and denatured aldolase B (Reznick et al., 1985). Their anti-denatured
aldolase B failed to immunoprecipitate catalytically active enzyme, but it effectively
precipitated smaller peptides resulting from proteolysis. They also found that native
aldolase B antibody failed to precipitate proteolytic fragments, but efficiently pelleted
aldolase B activity (Reznick et al., 1985). The results reported in this study confirm the

69
hypothesis that native epitopes of aldolase B require three-dimensional conformations
that mask denatured epitopes.
The 40 kD unmodified aldolase B predominantly occurred in cytosol (Cy)
fractions with <10% in lysosome-enriched (ML) fractions (Fig. 3-3b). Consistent with
previous results (Fig. 1-3), ubiquitin-aldolase B conjugates (bands >40 kD) were
enriched in ML fractions with less occurring in Cy fractions (Fig. 3-3b). Taken together,
the data suggest that ubiquitination can provide a mechanism for maintaining aldolase B
in a denatured conformation. This could contribute to enhanced degradation of aldolase
B by making degradative signals more accessible or by making the protein more
vulnerable to proteases.
Heat Stress-Induced Delivery of Aldolase A to Lvsosomes Requires Ubiquitination
Above, ubiquitinated aldolase B was confirmed to contribute to ubiquitin
conjugates that are enriched in autophagic vacuoles and lysosomes during nutrient stress
(Figs. 1-3, 1-4, and 3-3). The results suggested a role for ubiquitination in autophagic
degradation of aldolase B. During heat stress, ubiquitin-dependent autophagic
degradation of long-lived proteins has been demonstrated in E36 Chinese hamster lung
cells (Gropper, et al., 1991; Handley-Gearhart, et al., 1994). Our results suggested that
aldolase B was a possible substrate for this mechanism. However, E36 cells express
endogenous aldolase A but not aldolase B (described later). Both aldolase A and B are
established substrates for autophagy (reviewed in Chapter 1), so aldolase A was
examined as a substrate for ubiquitin-mediated delivery to lysosomes during heat stress.

70
In addition to autophagy, temperature-dependent cytosolic proteolysis
contributes to increased protein degradation during heat stress (next chapter; Hough and
Rechsteiner, 1984). During autophagy, cytosolic proteins, like aldolase A, are
sequestered into organelles (autophagic vacuoles and lysosomes) that can be pelleted by
differential centrifugation. To measure effects specific for the autophagic pathway,
aldolase A activity associated with pelletable organelles was assayed.
To examine a role for ubiquitination in the stress-induced degradation of aldolase
A in lysosomes, a system developed by Schwartz and Ciechanover was utilized for
measuring ubiquitin-dependent degradation of long-lived proteins (Gropper et al., 1991).
During heat stress, E36 cells undergo enhanced autophagic degradation of long-lived
proteins. However, ts20 cells derived from E36 cells harbor a temperature-sensitive
mutation in ubiquitination. Heat stress is non-permissive for the mutation , so
ubiquitination and enhanced autophagic degradation is inhibited in ts20 cells. The
degradative phenotypes were confirmed and are presented at the end of this chapter.
FDP aldolase activity was used to follow endogenous aldolase A, and acid
phosphatase activity was used to follow organelles in subcellular fractions collected by
differential centrifugation as described in Materials and Methods (Fig. 3-4).
Fractionation was optimized to maximize organelles released from cells, indicated by loss
of acid phosphatase from low-speed centrifugation pellets (LP), and to maximize
organellar integrity, indicated by fraction of acid phosphatase retained in high-speed
centrifugation pellets (HP). Acid phosphatase occurs as both an integral membrane

71
35%
0)
I 30%
O 25%
§ 20%
*5 15%
o 10%
£ 5%
LL.
0%
E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatment
70%
o
| 60%
O 50%
| 40%
l-
*5 30%
o 20%
£ 10%
u.
0%
Figure 3-4: Ubiquitin-Dependent Association of Endogenous Aldolase A with
Organelles. Aldolase (a.) and acid phosphatase (b.) reported as % total culture activity
(mean ± SD, n = 3 cultures) for subcellular fractions collected from E36 and ts20 cells
treated for 8.5 h as indicated; CT, control temperature; HS, heat stress; +CHQ, 80 pM
chloroquine; subcellular fractions were collected (Materials and Methods) and are
labeled only on the first set of three bars (E36, CT): LP, low-speed pellet (lOOOx g); HP,
high-speed pellet (100,000x g); Sum, total pelleted fractions (LP + HP); different from
CT, Student’s t-test: *, p <0.06; **, p <0.03; ***, p <0.009; ****, p <0.0008.
b. Acid Phosphatase Activity
Sum -r
fi ri
E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatment
a. Aldolase Activity

72
protein and as a soluble matrix protein inside lysosomes. For organelles isolated here,
acid phosphatase activity (about 60% of HP) was released by freeze-thaw (data not
shown), indicating that much of it was soluble in E36 cell lysosomes and served as an
adequate indicator of organellar integrity.
E36 cells were incubated at control (CT) and heat stress (HS) temperatures and
subcellular fractions pelleted during differential centrifugation were characterized for
aldolase and acid phosphatase enzymatic activities (Figs. 3-4 and 3-5). Relative to CT,
HS treatment significantly increased aldolase activity distributed in pelletable fractions
isolated from E36 cells (Fig. 3-4a). During 8.5 hour incubations that were used,
partitioning of aldolase to pelletable compartments had to be faster than loss. This is
consistent with accumulation of nascent autophagic vacuoles peaking by 6 hours after
autophagic induction (Lawrence and Brown, 1992). Chloroquine (+CHQ) caused a
more significant increase in pelletable aldolase activity. The effect of chloroquine
suggests that lysosomal degradation contributes to aldolase A flux out of pelletable
organelles consistent with an autophagic mechanism. In support of this, accumulation
caused by heat stress and chloroquine (Fig. 3-5, HS+CHQ) corresponds to a 1.5±0.3%/h
(mean ± SD, n = 3) increase in the sequestration rate for aldolase A which was similar in
magnitude to induced autophagic degradation for total long-lived proteins (data
presented in a later section). The results support endogenous aldolase A of E36 cells
undergoing autophagic delivery to lysosomes during heat stress.

73
3.0
a>
n o 05
+* â– 
re T-
£
a
(0
o
II
Q.
X
2.0
Q.
2
o
<
k.
a>
0.
o
(0
re
O
T3
O
o 1.5
1.0
T3
a>
N
re
E
o
— 0.5
0.0
Aldolase Accumulation
E36 E36 E36 ts20 ts20
CT HS HS+CHQ CT HS
Cell Line and Treatment
Figure 3-5: Ubiquitination Mediates Lysosomal Accumulation of Aldolase A During
Heat Stress. Using enzyme activities collected for Figure 3-4, aldolase was divided by
acid phosphatase to indicate relative aldolase associated with organelles (mean ± SEM, n
= 3); values were normalized to the 100,000 x g pellet (HP) of control temperature (CT)
to reflect aldolase accumulation relative to unstressed conditions. Labels are as in Figure
3-4. Student’s t-test: **, p <0.03; ***, p <0.009.
Heat stress (HS) by itself failed to affect the distribution acid phosphatase activity
in subcellular fractions (Fig. 3-4b). This indicated that increase of aldolase A in pellets
was not due to redistribution of lysosomal organelles and supported the idea that the
aldolase A was undergoing enhanced accumulation. Chloroquine treatment (+CHQ) had
no effect on total pelletable acid phosphatase (Sum) but caused a redistribution of acid
phosphatase from high-speed pellets (HP) to low-speed pellets (LP). It is known that

74
chloroquine treatment causes lysosomes to swell (Glaumann, et al., 1986). As a weak
base, chloroquine accumulates in organelles proportional to their acidity, and mature
lysosomes are the most acidic organelles. The results here indicate that some lysosomes
became large enough to pellet at lower centrifugation speeds.
A basic result of subcellular fractionation is that different pelleted fractions have
different contents of organelles (Rickwood, 1992). To demonstrate that aldolase A
accumulates in a subpopulation of organelles (presumably lysosomes), aldolase activity
was normalized to acid phosphatase activity and calculated the accumulation of aldolase
A in HP and LP relative to lysosomal content (Fig. 3-5). Significant accumulation of
aldolase activity only occurred in HP fractions during heat stress. In the presence of
chloroquine (+CHQ), there was a greater than two-fold accumulation of aldolase activity
in HP fractions but not LP fractions. The results suggest that aldolase A containing
organelles were preferentially isolated in HP even during CHQ treatment, and are
consistent with heat stress causing accumulation of aldolase A in a subpopulation of
lysosomes.
A previous study has shown that the fractional volume of autophagic vacuoles
and lysosomes does not significantly increase in heat stressed E36 cells (Lenk, et al.,
1992). Together, the data indicate that heat stress increases the flux of aldolase A into
autophagic vacuoles during heat stress. Unlike wildtype E36 cells, heat stress-induced
accumulation of aldolase activity with pelletable organelles failed to occur for mutant
ts20 cells (Figs. 3-4 and 3-5). Since heat stress inhibits ubiquitination in ts20 cells, this

75
suggested that aldolase A accumulation in organelles requires ubiquitination. The data
support a role for ubiquitination in heat-stress induced sequestration of aldolase A.
Using electron microscopic morphometry, a previous study shows that in heat stressed
ts20 cells conversion of autophagic lysosomes into residual bodies is specifically
inhibited, resulting in a 6-fold accumulation of lysosomal volume (Lenk, et al., 1992).
The subcellular fractionation results here indicate that earlier events in autophagic
degradation (aldolase A sequestration) might also involve ubiquitination. In conclusion,
the endogenous aldolase A of E36 cells appears to require ubiquitination for heat-stress
induced delivery to lysosomes.
Heat Stress-Induced Lysosomal Proteolysis of Aldolase B Requires Ubiquitination
Earlier in this chapter, ubiquitinated aldolase B in liver was shown to contribute
to ubiquitin conjugates that are enriched in autophagic organelles during starvation-
induced autophagy. Ubiquitination was required for heat stress-induced delivery of
endogenous aldolase A to lysosomes of E36 cells, suggesting that aldolase A was
degraded via ubiquitin-mediated autophagy. To examine whether aldolase B undergoes
ubiquitin-mediated autophagy like aldolase A, subcellular fractionation studies in the last
section were repeated with E36 and ts20 cells expressing epitope-tagged aldolase B
(RABM).
E36 and ts20 cells were transfected and selected for permanent expression of rat
aldolase B with the 9E10 myc epitope on its carboxyl terminus (RABM). The 9E10
epitope allowed efficient immunoprecipitation needed for degradation assays and

Figure 3-6: Transient Expression of RABM. a) E36 cells and b) ts20 cells transiently
expressing rat aldolase B with a carboxyl terminal myc tag (RABM) were processed for
immunofluorescence microscopy (Materials and Methods) and labeled with antibody
against aldolase B (ALDB) or the myc epitope (9E10). Phase contrast (Ph) images for
corresponding fields are shown below each immunofluorescent micrograph. Scale bar =
50 pM.

77
unambiguous identification of the exogenous aldolase B, RABM. In Figure 3-6,
transient expression of RABM was easily detected in a small fraction of cells with
antibody to either the myc epitope (9E10) or to native aldolase B (ALDB). Many
unlabeled cells indicated that E36 and ts20 cells do not express endogenous aldolase B.
Cell lines were isolated and screened for permanent RABM expression, and the highest
expressing lines for E36 and ts20 cells were designated E36AB and ts20AB, respectively
(Figs. 3-7 and 3-8).
After clonal selection all cells in a microscopic field were labeled for RABM in
permanent cell lines. Though most cells were brightly labeled, some were only dimly
labeled. Such labeling remained constant after multiple culture passages and for different
cell lines, suggesting that the variability was a trivial artifact of the immunofluorescence
protocol. According to immunofluorescence and western blot assays, different cell lines
had characteristic RABM levels that were maintained after multiple passages (data not
shown). Control experiments performed with cell lines expressing 5 to 10 fold
differences in RABM levels gave similar results. To facilitate detection, the highest
expressing lines (E36AB and ts20AB) were used extensively, and data are reported for
these lines. Immunofluorescent morphology indicated that RABM predominated in the
cytosol as shown by the presence of negatively labeled nuclei and vacuoles, providing
evidence that the recombinant protein demonstrated normal localization (Figs. 3-6, 3-7,
and 3-8).

78
E36 E36AB
Figure 3-7: Permanent Expression of RABM in E36 Cells. Transiently transfected E36
cells were selected and screened for permanent RABM expression; the highest
expressing cell line (E36AB) and untransfected cells (E36) were processed for
immunofluorescent detection as described in Materials and Methods with anti-9E10 myc
epitope (9E10, upper panels); phase contrast for corresponding fields are shown (Phase,
lower panels). Scale bar = 50 pM.

79
ts20 ts20AB
Figure 3-8: Permanent Expression of RABM in ts20 Cells. Transiently transfected ts20
cells were selected and screened for permanent RABM expression; the highest
expressing cell line (ts20AB) and untransfected cells (ts20) were processed for
immunofluorescent detection as described in Materials and Methods with anti-9E10 myc
epitope (9E10, upper panels); phase contrast for corresponding fields are shown (Phase,
lower panels). Scale bar = 50 pM.

80
41.3
40.0
41.3
40.0
Figure 3-9: Biochemical Detection of RABM Expression. Three confluent cultures (1,
2, 3) for each of the indicated cell lines was trypsinized, pelleted, suspended in 2 x
sample buffer, boiled, divided into two aliquots, run on duplicate SDS-PAGE gels,
western blotted, and duplicated blots were stained for aldolase B (upper panel) or the
9E10 myc epitope (lower panel); E36, original Chinese hamster lung cell line; ts20, an
E36-derived ts-mutant in ubiquitination; E36AB; an E36-derived cell line permanently
expressing RABM; ts20AB, a ts20-derived cell line permanently expressing RABM.
Arrow heads mark molecular weights (kD) for a doublet detected with anti-aldolase B.

81
On western blots of whole cells labeled with anti-aldolase B (Fig. 3-9), a single
faint 40 kD band was detected in untransfected cells (E36 and ts20), consistent with
cross-reactivity to aldolase A (Fig. 3-2b). Lysates of E36 and ts20 cells had aldolase
cleavage activity 35 to 40 fold higher for fructose 1,6-diphosphate than for ffuctose-1-
phosphate (data not shown). This difference for the two aldolase substrates is
characteristic for aldolase A, identifying this isoform as the prevalent endogenous
aldolase of E36 cells; note, aldolase B was not detected by immunofluorescence (Fig. 3-
6).
For RABM-expressing cells (E36AB and ts20AB), a closely spaced doublet of
bands was labeled with aldolase B antisera on western blots of whole cells (Fig. 3-9).
One band occurred at 40 kD coincident with endogenous aldolase A of untransfected
cells (E36 and ts20). A second strongly labeled band occurred at SDS-PAGE mobility
corresponding to 41.3 ± 0.1 kD (mean ± SEM, n = 6). On duplicate blots labeled for the
C-terminal myc epitope of RABM (9E10), long luminographic exposure times only
showed the 41.3 kD band (Fig.3-9). 41.3 kD matched the predicted molecular weight of
RABM, confirming that E36AB and ts20AB cells express full-length RABM.
If differences between immunoreactivities for hamster aldolase A (E36 cell
endogenous) and rat aldolase B (RABM) are similar to differences between titered
immunoreactivities (Fig. 3-2b) for purified rabbit aldolase A (Sigma) and purified human
aldolase B (Fig. 3-1), then immunoreactivities can be used to estimate RABM expression
relative to endogenous aldolase A. Endogenous aldolase A immunoreactivity was below

82
the linear range of western blot ECL assays in sample sizes subsaturating for aldolase B
detection. Immunodetection in the low range variably underestimated aldolase A by 3 to
>5 fold (data not shown), but allowed an upper limit for relative RABM expression to be
estimated. Estimates varied in the range of 0.2-2.4 RABM per endogenous aldolase A.
Though not precise, these overestimates indicated that RABM levels in permanent lines
were equal or less than the endogenous.
To determine whether RABM undergoes heat stress-induced autophagy, cultures
of E36AB and ts20AB were incubated at control (CT) and heat stress (HS) temperatures
and fractionated as done for untransfected cells in the previous section titled, “Heat
Stress-Induced Autophagy of Aldolase A Requires Ubiquitination.” Subcellular
distributions for activities of aldolase A and acid phosphatase were indistinguishable
between parental and RABM-expressing cell lines. The enzymatic activity for aldolase A
is ~10 fold more than aldolase B (Penhoet and Rutter, 1975). Given this, aldolase B
enzymatic activity was too low to detect above endogenous aldolase expression in E36
cells. To overcome this problem, aldolase B immunoreactivity was followed on western
blots of subcellular fractions (Fig. 3-10).
Figure 3-10a compares the distribution of immunoreactivities for RABM
(Aldolase B) and an integral membrane protein of lysosomes (LAMP2b) in subcellular
fractions isolated from E36AB cells incubated at control temperatures (CT). Details of
the subcellular fractionation are described in Materials and Methods. When cell sheets
were scraped from culture dishes and pelleted, some soluble proteins leaked out of the
cells, as indicated by the presence of RABM (Aldolase B) in the supernatant (Sc, Fig. 3-

Figure 3-10: RABM Associated With Lysosomes Undergoes Ubiquitin-Mediated
Proteolysis. Confluent E36AB and ts20AB cell cultures were incubated for 8.5 total
hours at indicated conditions and then fractionated as described in Materials and
Methods; samples of fractions were separated by SDS-PAGE, western blotted and
labeled for distribution of lysosomal membranes on higher molecular weight half of blots
(upper panels) and RABM on lower half of blots (lower panels) using antibodies to an
integral membrane protein of lysosomes (LAMP2b) or to native aldolase B (Aldolase B);
a) E36AB cells incubated in CT (see below) conditions were used to show distributions
for lysosomal membranes and RABM which were comparable for all treatments within
the variability of ECL detection (Amersham), 1% total culture equivalent of fractions
was loaded per lane except HP (lane 6) which used 10%: Sc, supernatant from cells
scraped then pelleted at 1000 x g; Ho, homogenate of lysed cell pellet; LP & Lsu, pellet
& supernatant from 1000 x g of Ho; HP & Hsu, pellet & supernatant from 100,000 x g
of Lsu; b) HP fractions isolated from cells incubated under indicated conditions were
loaded for equal acid phosphatase activity to show relative content of full-length RABM
(Aldolase B, 41.3), proteolyzed RABM (Aldolase B, 40.0), and lysosomal membranes
(LAMP2b): Co, control temperature, in DMEM + 10% FBS (normal culturing
conditions); CT, control temperature in MEM medium (experimental control); HS, heat
stress in MEM medium; +CHQ, medium supplemented with 80 pM chloroquine.
Simplified Diagram of Fractions*:
Cell Sheet —> scrape & 1000 x g —> sup = Sc
I
pel, homogenize = Ho
i
1000 x g —> sup = Lsu —> 100,000 x g —> sup =
Hsu
i i
pel = LP pel = HP
*sup, supernatants; pel, pellets

Aldolase B LAMP2b P* Aldolase B LAMP2b
84
a. E36ABCT
Sc Ho LP Lsu Hsu HP
1 2 3 4 5 6
HP Fractions
E36AB ts20AB
HS+
m*•
-41.3
^40.0
41.3
40.0

85
10a, lane 1). Membrane-bound organelles were retained in cells as indicated by the lack
of LAMP2b label in Sc.
The cell pellet was lysed, homogenized (Ho, Fig. 3-10a, lane 2), and used to
produce 1000 x g (low speed) pellet (LP) and supernatant (Lsu). Low speed pellets
generally contain nuclei, large cell fragments, and unbroken cells (Rickwood, 1992). LP
contained no detectable RABM, indicating that most cells were broken enough to lose
soluble proteins to Lsu (Fig. 3-10, lanes 3 and 4). Supporting this, LP contained about
15% of the aldolase A activity contained in Lsu (data not shown). The fact that some
aldolase A (Fig. 3-4a) but no RABM was detected in LP is probably due to the greater
affinity of aldolase A for pelletable cell components compared to aldolase B (Kusakabe,
et al., 1997). LAMP2b was approximately equally distributed in LP and Lsu, indicating
that about half the organelles (at least lysosomes) cofractionated with large cell
fragments or nuclei. In agreement with this, acid phosphatase activity was similarly
distributed between LP and Lsu (data not shown).
The Lsu was then used to produce 100,000 x g (high speed) pellet (HP) and
supernatant (Hsu). Such high speed centrifugations pellet all membrane-bound
organelles and leave soluble cytosolic (and leaked organellar) components in the
supernatant (Rickwood, 1992). Consistent with this, all the detectable lysosomal
membranes (Fig. 3-10a, LAMP2b) in Lsu (lane 4) were pelleted out of Hsu (lane 5). To
make RABM (Aldolase B) labeling in HP (Figure 3-10a, lane 6) comparable to that
loaded in lanes containing cytosol, 10 fold more HP equivalent was loaded.

86
On western blots of directly harvested whole cells (E36AB and ts20AB), aldolase
B immunoreactivity (RABM) primarily occurred at 41.3 kD (Fig. 3-9). In subcellular
fractions, a large proportion (>40%) of aldolase B immunoreactivity occurred at 40 kD
(Fig. 3-10a). This indicated that 41.3 kD RABM was proteolyzed to ~40 kD size during
fractionation. Like RABM, the LAMP2b-reactive protein was also proteolyzed as
indicated by the presence of a smear below its band on western blots (Fig. 3-10a,
especially visible in 10X loaded HP, lane 6). EDTA and storage on ice was used to
reduce protein degradation in subcellular fractionations but was insufficient to prevent
this presumably artifactual proteolysis. However, this result suggested that processing of
RABM from 41.3 kD to 40 kD could be used as an indicator of proteolysis.
Consistent for all treatments and fractions containing RABM (except HP), the
artifactual proteolysis was limited to processing 40-60% of the RABM (Fig. 3-10a, lanes
1, 2, 4, and 5). Only in HP fractions, processing of 41.3 kD RABM to 40 kD was
complete or nearly complete, such that aldolase B immunoreactivity collapsed from a
doublet to a single band at 40 kD (Fig. 3-10a, lane 6; Fig. 3-10b, lanes 1, 2, 3, 5, and 6).
However, if lysosomal degradation was inhibited by chloroquine (E36AB, HS+CHQ) or
autophagic degradation blocked by the non-permissive ubiquitination of heat stressed
ts20 cells (ts20AB, HS) then the complete processing of RABM in HP fractions was
blocked, as indicated by the persistence of 41.3 kD RABM in a doublet (Fig. 3-10b,
lanes 4 and 7). These data indicated that RABM proteolysis occurred in lysosomes
(CHQ-sensitive) and required ubiquitination (blocked in HS ts20AB). Whether greater

87
proteolysis of RABM seen in HP occurred in cells or during fractionation was not
determined. With the data from the last section, the results suggest that RABM
(aldolase B), like endogenous aldolase A, utilizes lysosomal degradation that requires
ubiquitination in E36 cells.
To demonstrate the presence of ubiquitinated aldolase B in E36 cells expressing
RABM, E36AB and ts20AB cell cultures were incubated at the different conditions used
for subcellular fractionation studies above. Then RABM protein was isolated using
9E10-specific immunoprecipitation and separation on SDS-PAGE (Materials and
Methods). Gels were western blotted and detected with native (N) and denatured (D)
aldolase B antisera. Since antisera for denatured aldolase B were most sensitive and
specific for ubiquitinated aldolase B on western blots (Fig. 3-2a and 3-3), these
antibodies were used to probe for ubiquitinated aldolase B (Fig. 3-1 la, upper panel). A
major stable ubiquitin-aldolase B conjugate occurred at 68 kD consistent with Ub68
found in rat liver (Fig. 1-3 and 3-3) and in Fao hepatoma cells (Fig. 1-4). This confirmed
that E36AB and ts20AB cells contained ubiquitinated aldolase B, including Ub68.
Similar levels of Ub68 were detected in all conditions, including heat stressed ts20 cells
(ts20AB, HS MEM) in which ubiquitination is inhibited. Unchanged ubiquitinated
protein under conditions of inhibited ubiquitination appears contradictory. Though
greatly inhibited, a low level of ubiquitination continues in heat stressed ts20 cells, but
this low level is insufficient to mediate cellular processes (Hischberg and Marcus, 1982;
Kulka, et al., 1988; Gropper, et al., 1991). The results here suggest that low levels of
ubiquitination were sufficient to maintain multiubiquitinated intermediates of aldolase B

Figure 3-11: Ubiquitinated Aldolase B Occurs in E36 and ts20 Cells Expressing RABM
Replicate sets of E36AB and ts20AB cultures were treated with indicated media
(DMEM +FBS or MEM) and temperatures (CT or HS), harvested, and
immunoprecipitated with 9E10 antibody; the immunoprecipitate was pelleted (P) from
the lysate and proteins remaining in the supernatant were precipitated with
trichloroacetic acid (S); P and S samples were boiled in 2 x sample buffer, split in equal
aliquots, and duplicate gels run on SDS-PAGE; a) Western blots to detect RABM
immunoreactivity (Aldolase B) were made from one gel and upper and lower portions of
blots were immunodetected with anti-denatured (upper panel, D) and anti-native (lower
panel, N) aldolase B, respectively; b) the duplicate gel was Coomassie stained, lane
numbers at the top of the Coomassie gel correspond to identical samples in lanes
numbered at the bottom of the blots in (a ); MW, molecular weight markers; molecular
weights (kD) are indicated at right.

Coomassie . Aldolase B
a E36AB ts20AB
CT HS CT HS
DMEM+FBS MEM MEM DMEM+FBS MEM MEM
PSPSPSPSPS PS
i
89
â—„ 68
41.3
40.0
â—„ 66.2
â—„ 55.0
â—„ 42.7
â—„ 40.0
â—„ 31.0
â—„ 21.5

90
(Ub68 probably contains 4 ubiquitins), but were insufficient to mediate the proteolysis of
RABM detected in HP fractions (Fig. 3-10b, lane 7). The occurrence of Ub68 in
different samples from different cell types (Figs. 1-3, 1-4, and 3-11) supports it as a
stable basal intermediate that probably requires more ubiquitination to facilitate
proteolysis. Alternatively, heat stress-induced ubiquitination could operate on the
machinery of stress-induced degradation. Further experiments are needed to distinguish
these alternatives.
As detected with antiserum to native aldolase B (N), immunoprecipitation with
the 9E10 epitope (specific for RABM) effectively pelleted all detectable 41.3 kD RABM
protein and more than half of a dim-labeled 40 kD protein, presumably endogenous
aldolase A (Fig. 3-1 la, lower panel). The 40 kD aldolase A was shown to lack 9E10
immunoreactivity (Fig. 3-9), indicating that RABM and endogenous aldolase A occur as
a complex in E36AB and ts20AB cells. This is consistent with the known tetrameric
structure of all FDP aldolase isozymes, wherein different subunits randomly and stably
oligomerize during synthesis (reviewed in Chapter 1). Ubiquitinated forms of aldolase B
were also removed from lysates by 9E10 immunoprecipitation (Fig. 3-1 la, upper panel),
suggesting that ubiquitinated RABM retained its C-terminal epitope tag or retained
associations with unmodified 9E10-immuno-reactive RABM subunits. If ubiquitinated
RABM is not associated with other aldolase subunits, then this would provide evidence
that ubiquitination might disassemble quaternary structure of aldolase B perhaps as an
early step in the degradative pathway. However, this possibility was not pursued here.

91
Experimental
Treatment
Association
With
Organelles
Limited
Early Lysosomal Late
Intermediates Proteolysis Intermediates
A.
Control
basal Aldolase A ^ 40 kD RABM
(trace, 41.3 kD RABM)
B.
Heat Stress
Aldolase A
(trace, 41.3 kD RABM)
40 kD RABM
c.
Heat Stress
+chloroquine
{}-
ft
Aldolase A
41.3 kD RABM
40 kD RABM
D.
Heat Stress
ts20 mutant
basal Aldolase A
41.3 kD RABM
40 kD RABM
Figure 3-12: Summary of Association and Limited Proteolysis of RABM and
Endogenous Aldolase A in Pelleted Organelles of E36 Cells. Each pathway corresponds
to experimental treatment described in boxes at left and correspond to the following
abbreviations used above: A) E36AB or ts20AB, CT; B) E36AB, HS; C) E36AB, HS
+ CHQ; D) ts20AB, HS. Since aldolase A was detected by enzymatic activity and is
inactivated by limited proteolysis, no late intermediates of Aldolase A are shown. Since
RABM detected on western blots shifts from 41.3 kD to 40 kD forms by limited
proteolysis, these forms are listed for early and late intermediates, respectively. Weight
of white vertical arrows indicate relative increases in detected levels of aldolase A and
RABM pelleted with organelles. Weight of black horizontal arrows indicate relative
rates proposed for processes listed in the heading.
Figure 3-12 summarizes the results of subcellular fractionation studies for
endogenous aldolase A and RABM expressed in E36 cells. Aldolase A activity is very
sensitive to proteolytic inactivation and loses 98% of its activity upon limited proteolysis
(Penhoet and Rutter, 1975; Horecker, et al., 1985). Since this made proteolyzed

92
aldolase A undetectable in the background of active aldolase A from E36 cells, aldolase
A activity was used to demonstrate association with organelles but not for detecting
proteolyzed intermediates of degradation. RABM was detected by western blotting with
antiserum to native aldolase B, allowing detection of limited proteolysis (41.3 kD —> 40
kD) products referred to here as “late intermediates” (Fig. 3-12, last column). Smaller
molecular weight intermediates of proteolysis were not detected, because they are not
recognized by anti-native aldolase B and probably are more rapidly degraded than 40 kD
aldolase B (Reznick, et al., 1985; Horecker, et al., 1985).
Under control conditions (Fig. 3-12, A), basal levels of aldolase A, 40 kD
RABM, and a trace of 41.3 kD RABM were detected in pelletable organelles. Heat
stress (Fig. 3-12, B) caused a partial increase in aldolase A activity with little apparent
change in RABM forms. However, a partial increase in trace levels of 41.3 kD RABM
were likely to be missed, because they were below the threshold for optimal Enhanced
Chemiluminescent detection (Amersham). Consistent with reaching the threshold for
detection, chloroquine inhibition of limited proteolysis caused a sudden signal increase in
41.3 kD RABM (Fig. 3-12, C). Chloroquine also caused an even more aldolase A to
accumulate. The results indicated that lysosomal proteolysis mediates loss of aldolase A
and aldolase B (RABM) associated with organelles, demonstrating sequestration of these
proteins into lysosomes.
When ubiquitination was inhibited by the ts20 mutation (Fig. 3-12, D) different
results were obtained for aldolase A and aldolase B (RABM). Consistent with

93
ubiquitination mediating degradative mechanisms after sequestration (Lenk, et al., 1992),
41.3 kD RABM accumulated in lysosomes. Under the same conditions, aldolase A
activity did not accumulate. For this to happen, reduced ubiquitination in ts20 cells
would have to increase lysosomal proteolysis or decrease sequestration of aldolase A
into lysosomes. Since the former possibility is unlikely, aldolase A probably requires
ubiquitination for sequestration as well as proteolysis. Since assays for aldolase A and
RABM were fundamentally different, the difference in ubiquitination effects between the
two isoforms might be due to differences in detection by enzymatic activity versus
immunoreactivity. Further experiments are needed to address this problem, but the
results here suggest that both aldolase A and aldolase B require ubiquitination for
degradation in lysosomes.
Ubiquitin-Mediated Autophagic Degradation Occurs in E36AB Cells
Above, evidence supporting ubiquitin-mediated proteolysis of aldolase B
(RABM) in lysosomes during heat stress was presented. To confirm that ubiquitin-
mediated autophagic degradation occurred during the fractionation studies above,
degradation rates of total long-lived proteins were measured for E36AB and ts20AB
cells under the same conditions. All protein degradation rates reported in this
dissertation are calculated as for degradative rate constants. Procedures are described in
Figure 3-13 and Materials and Methods. Arbitrary examples of regression analysis for
basal (control) and induced (stress) degradation rates are indicated in Figure 3-13. The
degradative rate constant, kd, is defined as the negative slope (coefficient of x) of the

Figure 3-13: Quantification of Degradation Rates, a) Cells were metabolically
radiolabeled and chased as described in Materials and Methods. Radiolabel signal (less
background) in TCA precipitates (for total long-lived proteins) or at appropriate
molecular weights for immunoprecipitates run on SDS-PAGE (for aldolase B) was
measured and magnitudes were natural-log transformed and plotted as described in
Materials and Methods. Regression lines, equations, and R2 correlation statistics are
shown for arbitrary examples of basal (Control) and heat stressed (Stress) protein
degradation, b) Slopes reported as decimal numbers in (a) are plotted as bars and
reported with units of %/h (= kd (/h) x 100%). The SEM of the slope was calculated
using Microsoft Excel Spreadsheet Analysis package: ANOVA regression error
(standard error of y-values at given x-values divided by the square root of the deviations
squared of x-values).

95
Linear (Control) Linear (Stress)
â–¡ control m stress
* w.w Yo
~ 3.0%
3 2.5%
(0
O'
2.0%
a>
>
re
1 1.5%
at
a>
E 1.0%
re
c
•5 0.5%
o
2
“■ 0.0%
Time Course as a Bar,
Negative Slope
+/- Slope Error

96
lines plotted in Figure 3-13a and of signal decrease in this dissertation are calculated as
done for Iq. Note of caution: different methods of calculating degradation are used by
different investigators which would give different rate magnitudes for the same data;
rates presented here are based on the empirically supported assumption that protein
degradation mechanisms operate with first order kinetics.
In this chapter, degradation rates are presented for total trichloroacetic acid
(TCA) precipitated proteins. Such rates represent an average of the pooled degradative
rate constants (kj) for all radiolabeled proteins over the time interval of the assay.
Variation in metabolic radiolabeling and chase intervals yield differentially labeled total
proteins due to widely different turnover rates for individual proteins, and such variation
would change the pooled kd. These factors were controlled to give comparable rates
between different experiments throughout this dissertation.
To confirmed that E36 cells undergo heat stress enhanced degradation of total
protein that requires ubiquitination, E36 cells, ts20 cells, and ts20El cells were
radiolabeled then chased under control (CT) and heat stress (HS) conditions, and
degradation rates were calculated as described above (Fig. 3-13). E36 cells were found
to undergo about a two fold enhancement in the degradation of long-lived proteins (Fig.
3-14, E36). The ts20 cell line derived from E36 cells has a temperature-sensitive
mutation in El ubiquitin-activating enzyme, and the heat stress (HS) is non-permissive
for El function in ts20 cells, greatly inhibiting ubiquitination (Kulka, et al., 1988) and
ubiquitin-mediated protein degradation (Gropper, et al., 1991). Consistent with this, the

97
reported as the fractional degradative rate in Figure 3-13b. For consistency and to allow
direct comparisons of data, all rates HS-enhanced degradation of long-lived proteins was
blocked in ts20 cells (Fig. 3-14, ts20). Furthermore, the mutant degradative phenotype
was rescued in ts20 cells permanently expressing wild type human El (Fig. 3-14,
ts20El).
E36 ts20 ts20E1
Cell Line
Figure 3-14: Heat Stress-Induced Degradation of Long-lived Proteins in E36 Cells
Requires El-Mediated Ubiquitination. Cultures of parent (E36) cells, El-ubiquitination
mutant (ts20) cells, and mutant rescued with cDNA for wildtype human El (ts20El)
cells were assayed for degradation of total TCA precipitated long-lived proteins
radiolabeled with 14C-valine (Fig. 3-13, Materials and Methods) reported for the
indicated conditions (mean ± SEM, n = 9); CT, control temperature; HS, heat stress; *,
HS > CT (Student’s t-test, p < 0.000001).

98
The results demonstrate that heat stress-induced degradation in E36 cells is dependent
on ubiquitination mediated by El. The degradation of long-lived proteins in E36AB and
ts20AB cells was assayed (Fig. 3-15), and the results were similar to those for
untransfected E36 and ts20 cells (Fig. 3-14). This demonstrated that RABM expression
had no effect on the degradative phenotypes in these cells. Cultures were also treated
with the relatively specific inhibitor of autophagy, 3-methyladenine (3MA), or with the
lysosomal hydrolase inhibitor, chloroquine (CHQ). Both inhibitors effectively blocked
the enhanced degradation of long-lived proteins (Fig. 3-15). The results confirmed that
heat stress-induced degradation of most long-lived proteins occurred in lysosomes via
autophagy.
In Figure 3-15, ts20AB cells appear to have reduced basal degradation relative
to E36AB cells. This result was not consistent between experiments, and in general,
E36 and ts20 cells had similar basal degradation rates that were unaffected by RABM
expression (data not shown). Chloroquine (CHQ) and 3-methyladenine (3MA) inhibited
a fraction of basal protein degradation at the concentrations shown (Fig. 3-15). Though
the effect of chloroquine was marginal here (40 pM), at higher concentrations (80 & 160
pM) inhibition of basal degradation was more significant (data not shown). CHQ at the
higher concentrations and 3MA at the least inhibitory concentration reduced the viability
of ts20 cells during heat stress (Materials and Methods; data not shown), so degradation
measurements for such treatments were not included. As indicated by sensitivity to
CHQ or 3MA, 17±6% of basal degradation of long-lived proteins occurred by lysosomal
degradation via autophagy, corresponding to a rate of 0.3 ± 0.1%/h (mean ± SEM, n =

99
12). Given the estimated basal rate of autophagy, heat stress (HS) caused a calculated
6.6 ± 1.0 fold induction over basal autophagic degradation in uninhibited wildtype E36
E36AB E36AB E36AB ts20AB ts20AB
+CHQ +3MA +CHQ
Cell Line & Inhibitor
Figure 3-15: Heat Stress-Induced Autophagic Degradation of Long-lived Proteins that
Requires Ubiquitination Occurs in Cells Expressing RABM Cultures of E36AB and
ts20AB cells were metabolically labeled with 35 S-methionine, and at various times of
chase, cultures were lysed in modified RIPA buffer and 2% of the lysate was TCA
precipitated and processed to measure degradation of long-lived proteins (mean ± SEM,
n = 16-34) as previously described (Fig. 3-13 and Materials and Methods); CT, control
temperature; HS, heat stress, indicated chase medium were supplemented with 40 pM
chloroquine (+CHQ) or 5 mM 3-methyladenine (+3MA). Significant differences
(Student’s t-test, p < 0.05): mean < untreated CT in corresponding cells (*, p < 0.05;
**, p < 0.02; ***, p< 0.008); ****, E36AB, HS > CT (p <0.0002). The remaining
lysates (98% of the volume) were used to immunoprecipitate and measure the
proteolysis of RABM discussed below (Fig. 4-4).

100
cells (mean ± SEM, n = 3). In ubiquitination mutant ts20 cells, enhanced degradation
was blocked (Figs. 3-14 and 3-15; Gropper, et al., 1991), and a 6-fold increase in
fractional volume of autophagic vacuoles occurred (Lenk, et al., 1992). Together, the
data support a model in which intralysosomal complete degradation of proteins requires
ubiquitination, but sequestration does not (Lenk, et al., 1992).
Basal autophagic degradation explained why aldolase A and RABM were found
with pelletable organelles in all conditions (Fig. 3-4a and 3-10). Neither aldolase A
activity nor RABM immunoreactivity accumulated in HS ts20 cells, even though
autophagic sequestration was increased 6-fold (previous paragraph). Ubiquitination was
required for limited proteolysis of RABM from 41.3 kD to 40 kD (Fig. 3-10b), but other
limited proteolysis appeared sufficient enough to destroy enzyme activity and
immunoreactivity, preventing multifold accumulation of detectable aldolase. Though
detectable aldolase fragments did not accumulate, proteolysis did not seem to proceed to
amino acids, because enhanced lysosomal degradation of TCA-precipitable polypeptides
was blocked in HS ts20 cells (Figs. 3-14 and 3-15). The data are consistent with
ubiquitination being required for a subset of lysosomal protease activities needed to
completely degrade long-lived proteins to amino acids, and one such protease can be
detected as intralysosomal limited proteolysis of RABM (aldolase B).

CHAPTER 4:
TEMPERATURE MODULATES AUTOPAGY AND
CYTOSOLIC PROTEOLYSIS OF ALDOLASE B
Introduction
In the literature, starvation-induced autophagic degradation of aldolase B has
been proposed, and heat stress-induced autophagic degradation of long-lived proteins in
E36 cells was shown to require ubiquitination. As shown above, ubiquitinated aldolase
B (Ub68) occurred in cultured E36 cells and in vivo enriched with organelles of
starvation-induced autophagy isolated from rat liver. In E36 cells, assays for aldolase
associated with organelles showed that lysosomal accumulation of endogenous aldolase
A and lysosomal proteolysis of exogenous aldolase B (RABM) required ubiquitination.
The results are consistent with degradation of aldolase isoforms in general occurring by
ubiquitin-mediated autophagic degradation inside lysosomes. However, aldolase A and
aldolase B have been proposed to undergo limited proteolysis on the outside of
lysosomes independent of acidic pH (Pontremoli, et al., 1982; Horecker, et al., 1985;
Sygush, et al., 1990). To examine the contribution of cytosolic and lysosomal
mechanisms, rates for basal and heat stress-induced proteolysis of aldolase B (RABM) in
E36 cells were quantified in the presence of lysosomal inhibition. The results below
show that temperature-dependent cytosolic proteolysis of aldolase B was independent of
ubiquitination, that complete cytosolic protein degradation to amino acids was
101

102
independent of temperature, and that autophagic degradation also increased in a
temperature-dependent manner.
Ubiquitin-Independent Cytosolic Proteolysis of Aldolase B
To measure proteolysis of aldolase B, the decrease of radioactivity in RABM
protein bands on gels of 9E10-immunoprecipitates isolated from metabolically labeled
cells during pulse-chase experiments was measured. Immunoprecipitation was efficient
with anti-9E10 (Fig. 3-1 la, lower panel, N) and consistently yielded more RABM than
for anti-aldolase B (Fig. 4-1). 9E10 immunoprecipitation gave the most reproducible
data and was used extensively for experimental repetitions; unless otherwise stated,
degradation rates for RABM are reported for such data. Standard curves were done to
confirm a linear response for quantification of the radiolabeled RABM (Fig. 4-2).
Degradation rates were calculated in the same manner as for total long-lived protein
degradation using RABM-specific signal (Fig. 3-13). Since a specific protein was
measured, the resulting degradation rates were equivalent to the degradative rate
constant (kd) for RABM (aldolase B) proteolysis. The results below agree well with
published kd for aldolase B degradation in cultured cells (1.0-2.5%/h basal and 3.0-
5.0%/h stress-induced).
To determine the contribution of ubiquitination in heat stress-induced
degradation of aldolase B, degradation rates for RABM in E36AB and ts20AB cells at
control(CT) and heat stress (HS) temperatures were measured (Fig. 4-3). The

103
9E10 Aldolase B
E36AB ts20AB ts20AB ts20
Figure 4-1: Immunoprecipitation of RABM With Antibodies Against 9E10 or Native
Aldolase B Epitopes. Anti-myc-sepharose in mRIPA buffer lysates (9E10) or aldolase B
antiserum precipitated with protein A-agarose in SLB lysates (Aldolase B) were used to
immunoprecipitate radiolabeled RABM from E36AB and ts20AB cell lines; ts20 cells
lacking RABM demonstrate background for the least stringent immunoprecipitation
(Aldolase B). Arrowheads indicate only specifically precipitated bands (40 and 41.3
kD), migrating in a region of consistently low background. The prominent 44-45 kD
band in 9E10 immunoprecipitations was non-specific, representing actin that has a high
affinity for sepharose (Sigma technical support).

104
from RABM Expressing Cells
Figure 4-2: Standard Curve For 9E10 Immunoprecipitation of RABM. Confluent
cultures of ts20AB and ts20 (untransfected) cells were metabolically labeled with ?5S-
methionine, lysed in mRIPA buffer, lysates precleared, and immunoprecipitated (anti-
9E10 conjugated to sepharose) done as described in Materials and Methods, except
ts20AB lysate (containing maximal RABM) was diluted with ts20 lysate (lacking
RABM) as indicated. Each immunoprecipitation was done from -400,000 cell
equivalents of lysate. Regression line and equation for least squared deviation is shown
with R2 correlation statistic; R = 0.9637 (ideal would be a slope of 1.00 with intercept of
0.00 and R2=1.00).
degradation of RABM was two fold enhanced by heat stress. Though starvation-
induced degradation of aldolase B is established (reviewed in Chapter 1), this is the first
demonstration that aldolase B undergoes enhanced degradation during heat stress.
Furthermore, induced proteolysis continued even when ubiquitination was inhibited (Fig.
4-3 HS,ts20AB), indicating involvement a ubiquitin- independent mechanism. This
suggested that in addition to ubiquitin-mediated lysosomal proteolysis (Fig. 3-12), a

105
second proteolytic mechanism for RABM occurred at rates similar to heat stress-induced
autophagy that required ubiquitination (Fig. 3-15).
5%
E36AB ts20AB
Cell Line
Figure 4-3: Heat Stress-Induced Degradation of RABM Occurs Independently of El-
Mediated Ubiquitination. Indicated cell lines were metabolically labeled with 35S-
methionine, chased for various times, immunoprecipitated, radioactivity at the molecular
weight of RABM was quantified, and regression analysis performed to calculate the
degradative rate constant ± standard error (n = 31 to 34) for RABM as described in
Materials and Methods, under control (CT) and heat stress (HS) conditions. Reminder:
HS is non-permissive for El-mediated ubiquitination in ts20AB cells. For both cell lines,
RABM degradation at HS was higher than at CT (p < 0.0007).

Temperature-Dependent Cytosolic Proteolysis in Fao Cells
106
To determine how much heat stress-enhanced proteolysis of RABM occurred in
lysosomes, chloroquine (CHQ) was used to inhibit lysosomal hydrolases (Fig. 4-4).
E36AB E36AB E36AB ts20AB ts20AB
+CHQ +3MA +CHQ
Cell Line + Drug
Figure 4-4: Heat Stress-Induced Degradation of RABM Occurs Independently of
Lysosomal Hydrolasas and Autophagv. Cultures were treated and processed as for
Figure 4-18 to calculate degradation rates (mean ± SEM, n = 16 to 34), except cultures
were treated with either 5 mM 3-methyladenine (+3MA) or 40 pM chloroquine (+CHQ)
to inhibit autophagy or lysosomal hydrolases, respectively. Student t-tests: for all
treatments, HS > CT (p < 0.0013); *, 3MA partially inhibited HS degradation (p <
0.031); all other differences were not significant at a 0.05 level. Note: 2% of each
culture lysate was TCA precipitated and used to measure degradation of long-lived
proteins occurring under identical conditions (Fig. 3-15).

107
Chloroquine failed to reduce the heat stress-induced degradation rate for RABM; note,
the apparent increase shown in Fig. 4-4 was neither statistically significant nor
reproducible (data not shown). Even when ubiquitination and lysosomal degradation
were simultaneously inhibited (ts20AB+CHQ), enhanced degradation of RABM still
continued (Figs. 4-4). This indicated that heat stress-induced proteolysis of aldolase B
in E36 cells was independent of ubiquitin-mediated autophagy and lysosomal
degradation. Supporting this, 3-methyadenineonly marginally inhibited heat stress-
induced autophagic degradation (Fig. 4-4), and the marginal inhibition was accounted by
a comparable reduction of basal protein degradation observed for samples from the same
cultures (Fig. 3-15). These results support a role for cytosolic proteolysis in the heat
stress-induced degradation of aldolase B.
The inhibitor resistant proteolysis of RABM (Fig. 4-4) seemed to contradict the
inhibitor sensitive degradation of total long-lived proteins measured for the same cultures
(Fig. 3-15), suggesting that degradation of RABM occurred by a selective cytosolic
mechanism which was specific for a fraction of total long-lived proteins that includes
aldolase B. An alternative explanation is possible, because protein degradation
measurements using TCA and immunoprecipitation are fundamentally different from
each other. Degradation measurements for total proteins utilize TCA precipitation
which cannot distinguish full length protein from polypeptide fragments created by
partial proteolysis. However, immunoprecipitation is sensitive to limited proteolysis,
because a single proteolytic cleavage can disrupt the size and structure of a protein

108
removing it from an SDS-PAGE band used to quantify degradation. Furthermore,
aldolase B is known to undergo proteolysis to relatively large polypeptide fragments that
cannot be recognized by antibodies against the native protein (Reznick, et al., 1985),
demonstrating that partial proteolysis can disrupt immunoreactivity. Immunoreactivity
lost to partial proteolysis causes loss of radiolabel from corresponding
immunoprecipitates, including fragments that might be quite large. Thus,
immunoprecipitation effectively detects limited proteolysis whereas TCA precipitation
requires complete degradation to amino acids. Given this, cytosolic proteolysis of
aldolase B (RABM) occurred independently by partial proteolysis that produced
undetectable polypeptide fragments (TCA precipitable) requiring autophagic degradation
in lysosomes for complete proteolysis.
Since polypeptide fragments of aldolase B could not be detected the possibility
remains that aldolase B underwent an enhanced complete degradation in cytosol during
heat stress. However, this would have to be a unique pathway for aldolase B because
for most proteins (TCA precipitated) autophagic degradation was required. Given this, a
cytosolic mechanism for complete degradation of aldolase B would have to be selective
for aldolase B, requiring s recognition mechanism that is novel and unsubstantiated in the
literature. This seem less likely than a more general model in which many proteins,
including aldolase B, undergo partial cytosolic proteolysis followed by autophagic
degradation in lysosomes.

109
Degradation assays using anti-aldolase B immunoprecipitation gave similar
results to anti-9E10 (data not shown). This indicated that proteolysis of RABM was not
limited to the epitope tag but included at least some degradation of aldolase B protein.
Since immunoprecipitations were done from extracts of cell sheets, loss of RABM to the
media would look like degradation. To control for this, heat stress release of aldolase
activity into media was measured and found statistically insignificant compared to
variation in induced degradation measurements (Fig. 4-5).
aop/01
Ftelease to [Degradation
Medium
Measirerrert of Adolase
Figure 4-5: Heat Stress-Induced Degradation of Aldolase B was Not Due to Release of
Aldolase to Medium. During chase treatments, aliquots of media were assayed for
aldolase activity; after the final chase, cells were harvested, total activity in the culture
was calculated, and the fractional rate of activity loss calculated. The rate calculation
and measurements for the degradation of aldolase B are described in Materials and
Methods. Release of aldolase to media > 0 (p < 0.034) but had a low probability of
contributing an effect on degradation rates (Student’s t-test, p < 0.0001). At control
temperatures (data not shown), aldolase activity released to the medium was not
significantly greater than zero (p > 0.4).

110
Starvation-Induced Autophagic Degradation of Aldolase B in Fao Cells
To control for the nature of the epitope tag on RABM and determine whether
cytosolic proteolysis of aldolase B generally occurred in different cells, heat stress-
induced proteolysis of epitope-tagged human aldolase B (HAHAB) expressed in Fao
cells was examined (next section). HAHAB carried a 12CA5 HA (hemagglutinin)
epitope on its amino terminus. Measuring degradation of HAHAB in Fao cells versus
RABM in E36 cells controlled for cell type (rat hepatoma vs. Chinese hamster lung),
position and identity of the epitope tag (amino-HA vs. carboxyl-myc), and the source
species of the recombinant aldolase B (human vs. rat). Whether heat stress can induce
autophagy in Fao cells was not known. Before examining this possibility, it was
reasonable to confirm that starvation-induced autophagic degradation of exogenous
aldolase B (HAHAB) occurred like that for endogenous aldolase B.
Fao cells retain many characteristics of differentiated hepatocytes, including
expression of endogenous aldolase B (Deschatrette et al., 1979; Deschatrette and Weiss,
1974), and starvation-induced degradation of long-lived proteins (Fig. 4-6, bar 2).
Treatment of Fao cultures with 3-methyladnine (3MA) to block autophagic
sequestration, chloroquine (CHQ) to inhibit lysosomal acid hydrolases, or leupeptin
(LEUP) to inhibit lysosomal serine proteinases blocked starvation-induced degradation
(Fig. 4-6), demonstrating that enhanced degradation of long-lived proteins occurred in
lysosomes via autophagy in starved Fao cells as well as in heat stressed E36 cells. E36
cells did not undergo starvation-induced degradation (data not shown), but Fao cells

Ill
provided an opportunity to examine both starvation-induced and heat stress-induced
degradation of aldolase B in the same cell type.
c
o
TO
"O
CO
o>
0
Q
c
'0
o
L_
CL
T3
0
>
1
O)
c
o
Fed
Starved
Starved
+3MA
Starved
+LEUP
Starved
+CHQ
Figure 4-6: Starvation Induces Degradation of Long-lived Proteins in Fao Cells. Fao cell
cultures were metabolically labeled with 14C-valine, chased with DMEM + 10% FBS
(Fed, bar 1) or Krebs-Heinseleit Medium (Starved, bars 2-5) containing “cold” 10 mM
valine, and loss of TCA precipitable counts was used to calculate degradation rates
(mean ± SEM, n = 8-16) as described in Materials and Methods; Krebs-Heinseleit is an
amino acid free minimal medium; 3MA (bar 3), 10 mM 3-methyladenine; LEUP (bar 4),
300 mM leupeptin; CHQ (bar 5), 160 pM chloroquine. Significant differences
(Student’s t-test, p < 0.0001): bar 2 > bars 1, 3, 4, or 5; bar 5 < bar 1.

112
Fao FaoAB
Figure 4-7: Permanent Expression of HAHAB in Fao Cells. Fao cells were transfected
and selected for permanent expression of human aldolase B carrying a 12CA5 HA
epitope tag on its amino terminus (HAHAB), resulting a cell line permanently expressing
HAHAB (FaoAB). Untransfected Fao (Fao) and FaoAB cells were processed for
12CA5 immunofluorescence microscopy (upper panels) as described in Materials and
Methods; Phase (lower panels), phase contrast of same field as upper panels. Scale bar =
50 pM.

113
To effectively measure the degradation of aldolase B in Fao cells, an Fao cell line
permanently expressing epitope-tagged aldolase B (HAHAB) was isolated. Figure 4-7
shows the immunofluorescent signal for the HA epitope (12CA5) in a cell line
permanently expressing HAHAB (FaoAB). Note the bright signal in FaoAB cells
relative to the untransfected parent cells (Fao). Anti-HA epitope (12CA5) specifically
detected expression of tagged exogenous aldolase B as indicated by lack of label in
untransfected cells (Fig. 4-7, Fao). The presence of negatively labeled nuclei and
vacuoles supported a cytosolic localization for HAHAB. Consistent with Fao cells
expressing endogenous aldolase B, Fao and FaoAB cells stained with anti-aldolase B had
similar labeling patterns to each other (data not shown) which resembled the transfected
cells labeled with anti-12CA5 (Fig. 4-7, FaoAB).
Expression of HAHAB was confirmed on western blots (Fig. 4-8). Western blot
analysis with anti-aldolase B (Fig. 4-8a) showed 40 kD endogenous aldolase B in
untransfected Fao cells (Fao). FaoAB cells (FaoAB) also contained a 41.3 kD aldolase
B-immunoreactive protein indicating HAHAB expression. Non-specific background and
a 41.3 kD marker for epitope-tagged aldolase B were provided by ts20 and ts20AB cells
that lack endogenous aldolase B. HAHAB comigrated with RABM, consistent with
12CA5 and 9E10 epitope constructs being similar in size. Fao cells express over 10 fold
more aldolase A than aldolase B (Deschatrette et al., 1979; Deschatrette and Weiss,
1974), so the pattern of labeling was consistent with HAHAB expression equal to or less
than total endogenous aldolase (isoforms A + B).

114
b. 12CA5-IP
FaoAB Fao
Figure 4-8: Biochemical Detection of HAHAB. a) anti-aldolase B western blot analysis
was performed on whole cell extracts from the indicated cell lines as described in
Materials and Methods; ~1 cm2 culture area equivalent loaded per lane from different
confluency: ts20AB and ts20 lanes from tightly confluent, first FaoAB and last Fao lanes
from recently confluent, remaining lanes from subconfluent; ts20 cells have no
endogenous aldolase B, and ts20AB cells express a different epitope-tagged aldolase B,
RABM; b) autoradiograph of duplicate anti-12CA5 (HA epitope) immunoprecipitates
prepared from 5S-methionine labeled FaoAB and Fao cultures as described in Materials
and Methods. Molecular weights for proteins specifically detected by the methods are
indicated.

115
On autoradiographs of anti-12CA5 immunoprecipitates (12CA5-IP) from cells
metabolically labeled with radioactive amino acid, a doublet of HAHAB (41.3 kD) and
endogenous aldolase (40.0 kD) was specifically precipitated from FaoAB cells and not
Fao cells (4-8b). Since endogenous aldolase lacked 12CA5 immunoreactivity (Fig. 4-7,
Fao), the 40 kD aldolase subunits appeared to be associated with HAHAB for
immunoprecipitation. This provided evidence that HAHAB subunits tetramerized with
endogenous subunits. The results indicated that HAHAB was permanently expressed in
a manner expected for a subunit of aldolase. Furthermore, aldolase protein bands
migrated in a region of low background on SDS-PAGE of immunoprecipitates
facilitating quantification of radioactivity associated with HAHAB (Fig. 4-8b).
In order to compare stress-induced degradation of HAHAB in Fao cells to
previous results for RABM in E36 cells (Fig. 4-3 and 4-4), 12CA5-immunoprecipitation
was used to isolate and measure the degradation of HAHAB (Fig. 4-9). Starvation
increased the degradation of HAHAB by at least two fold (Figs. 4-9 and 4-11) which
was even greater than the enhancement of total long-lived protein degradation (Fig. 4-
6). Previously, heat stress-induced degradation of RABM (aldolase B) in E36 cells
resisted lysosomal inhibitors (Fig. 4-4), but starvation enhanced degradation of HAHAB
was completely inhibited by chloroquine (CHQ) or 3-methyladenine (3MA) in Fao cells
(Fig. 4-9). The data indicated that HAHAB was degraded in lysosomes via autophagy
during starvation, and that cytosolic proteolysis was constant at the constant temperature
(37°C) during starvation in E36 cells.

116
None +CHQ +3MA
Inhibitor Treatment
Figure 4-9: Starvation-Induced Autophagic Degradation of HAHAB in Fao Cells.
FaoAB cells labeled with 35S-methionine were chased with excess “cold” methionine in
DMEM + 10% FBS (Fed) or Krebs-Heinseleit medium without amino acids (Starved)
with 160 |iM chloroquine (+CHQ), 10 mM 3-methyladenine (+3MA), or no additions
(None); radiolabel in immunopurifed HAHAB was used to calculate degradation rate
(mean ± SEM, n = 11-23) as described in Materials and Methods; *, HS > CT,
Student’s t-test (p < 0.0005).
To confirm that HAHAB was representative of endogenous aldolase B,
starvation-induced loss of endogenous aldolase B was examined on western blots (Fig.
4-10). When protein synthesis is inhibited protein decreases reflect degradation rates
(Henell, et al., 1987). In fed confluent cultures (Fed), aldolase B levels remained
constant or slightly increased (statistically insignificant) during 8 hour incubations (Fig.

117
4-10). Starvation inhibited protein synthesis (data not shown), and endogenous aldolase
B levels decreased at a rate that was statistically indistinguishable from the enhanced
degradation of HAHAB (Fig. 4-9 and 4-10). 3MA or CHQ inhibited the loss of
endogenous aldolase B to a rate of ~2%/h (Fig. 4-10), consistent with basal cytosolic
degradation (Fig. 4-9) and indicating that endogenous aldolase B and HAHAB utilized
same starvation-induced autophagic mechanism for degradation.
Fed Starved Starved Starved
+3MA +CHQ
Figure 4-10: Starvation-Induced Autophagic Degradation of Endogenous Rat Aldolase
B in Fao Cells. Untransfected Fao cells were incubated in the chase media of Figure 4-9
(same labels) for various times, and whole cells directly harvested by boiling in sample
loading buffer were analyzed on anti-aldolase B western blots, and changes (mean ±
SEM, n = 16-24) in the level of aldolase B were calculated as previously described
(Materials and Methods). Rates of decrease were plotted, so negative value indicates
aldolase B increase. *Starved > Fed (Student’s t-test, p < 0.0001); **
Starved+Inhibitor < Starved (p < 0.0007); ***Fed = 0%/h (p > 0.15).

Temperature-Dependent Autophagv and Cytosolic Proteolysis
118
Due to the temperature-sensitive mutation in ts20 cells, E36 and ts20 cells were
routinely maintained at 30.5°C, according to established protocols used here (Gropper,
et al., 1991). For the established experimental system, control temperatures were 30.5°C
(CT) and heat stress was 41 5°C for one hour then 39.5°C (HS). Heat stress and
starvation at 37°C, both, enhanced the complete degradation of total TCA precipitated
polypeptides by autophagy (Figs. 3-15 and 4-6). In agreement with this, starvation
enhanced the autophagic degradation of immunodetected aldolase B at 37°C (Figs. 4-9
and 4-10). These results indicated that starvation-induced autophagic degradation of
aldolase B was rate limiting and faster than cytosolic proteolysis of aldolase B at 37°C in
Fao cells. In E36 cells, heat stress increased cytosolic proteolysis of immunoprecipitable
aldolase B to a rate greater than heat stress-induced autophagic degradation (Figs. 3-14,
3-15, 4-3, and 4-4). The cytosolic proteolysis, though probably partial, disrupted
immunoprecipitation of aldolase B such that degradation of aldolase B appeared to be
independent of lysosomal degradation.
To determine whether heat stress-induced cytosolic proteolysis of aldolase B was
a general phenomenon beyond E36 cells, the heat stress and starvation protocols were
compared using Fao cells (Fig. 4-11). Consistent with E36 cells, 39.5°C heat (stress)
caused increased proteolysis of HAHAB relative to 30.5°C control temperature
(unstressed) in Fao cells (Fig. 4-11, Heat Stress Induction, untreated). The proteolysis

119
enhanced by 39.5°C was resistant to chloroquine treatment (CHQ), indicating the
presence of the enhanced cytosolic mechanism (Fig. 4-11, Heat Stress Induction). Sister
untreated +CHQ untreated +CHQ
Heat Stress Induction Starvation Induction
Experimental System & Chloroquine (+CHQ)
Figure 4-11: Temperature Increases Cytosolic Proteolysis and Starvation Induces
Lysosomal Proteolysis of Aldolase B (HAHAB V Heat stress and starvation induction of
protein degradation in Fao cells and degradation of HAHAB was done as previously
described above (mean ± SEM, n = 14-16). “Control” and “Stress” treatments are
different for the two induction systems. The degradation rates are estimates of kj for
HAHAB proteolysis. Student’s t-test: *, Stress > Control (p < 0.001). Arrows connect
temperatures that indicate kd estimates for “cytosolic” (CHQ-resistant) proteolysis.
cultures of the Fao cells confirmed that starvation-induced autophagic degradation of
aldolase B occurred at 37°C when amino acids and additional nutrients were withheld
(Fig. 4-11, Starvation Induction). For the starvation-induced degradation at 37°C, it

120
was confirmed that inducible lysosomal degradation was inhibited by chloroquine
treatment (Fig. 4-11, Starvation Induction, ±CHQ). Altogether, the data demonstrated
that cytosolic proteolysis of aldolase B can occur in different cell types (E36 Chinese
hamster lung and Fao rat hepatoma) with different aldolase B constructs (RABM and
HAHAB), and is temperature-dependent.
The degradation measurements for aldolase B represent rate constants (kj) set by
rate-limiting mechanisms. For cytosolic proteolysis of HAHAB, the rate limiting step
increased proportionally with increased temperature (Fig. 4-11, arrows and
temperatures). This suggested that increased reactivity was by direct thermal stimulation
of a rate-limiting cytosolic mechanism. To test this possibility, the k determined for three different temperatures (30.5°C, 37°C, and 39.5°C) were used to
make Arrhenius plots (Fig. 4-12) to test this possibility.
Evidence derived from the Arrhenius plots (Fig. 4-12) is limited by the fact that
only three temperatures were available. Total cellular degradation of long-lived proteins
in cultured smooth muscle cells was previously shown to undergo direct thermal
stimulation with Ea = 18 kcal/mole at 15-37°C (Bates, et al., 1982). With similar data
here (Total TCA), an Ea = 19.6 ±1.5 kcal/mole at 30.5-39.5°C was calculated and was
statistically indistinguishable (p > 0.1) from the previously published result (Fig. 4-12).
This argued that our calculations gave reasonable estimates of Ea.

121
Lyso
TCA
Cyto
HAHAB
Cyto
TCA
Total
TCA
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o'
o
o
o'
o
o
Reciprocal Temperature, 1/K
Figure 4-12: Heat Stress Induces Cytosolic Proteolysis of Aldolase B By Thermal
Stimulation of Lvsosomes. Cytosolic (Cyto, light unbroken line) or lysosomal (Lyso,
broken line) proteolysis was degradation resistant or sensitive, respectively, to inhibition
with 3-methyladenine or chloroquine; the sum of these (Cyto+Lyso) represents total
cellular proteolysis (Total, heavy unbroken line); degradation rates for immunopreci-
pitated aldolase B (HAHAB, filled symbols) or TCA precipitated polypeptides (TCA,
open symbols) were used as k d-estimates to construct Arrhenius plots with slope =
activation energy in kcal/mole (Ea), an arc connects estimated slope, Ea (mean ± SEM, n
= 7 or 8) accompanied by the corresponding correlation coefficient (r) for a least squares
linear plot; square represents cytosolic proteolysis of HAHAB; triangle, circle, or
diamond represents cytosolic, lysosomal, or total proteolysis of long-lived proteins,
respectively. By Student’s t-test, all slopes were much > 0 and different from each other
(p < 0.0004), except “Cyto TCA” was close but slightly > 0 (p < 0.028), and “Cyto
HAHAB” slope was indistinguishable from “Lyso TCA” (p > 0.53).

122
Based on sensitivity to inhibitors, lysosomal (Lyso) and cytosolic (Cyto) degradative
mechanisms for long-lived proteins were separated (Fig. 4-12, TCA). Both had linear
Arrhenius plots, giving Ea = 40.6 ±1.8 and 5.1 ± 2.2 kcal/mole for lysosomal and
cytosolic degradation, respectively. A plot for the cytosolic proteolysis of HAHAB
(aldolase B) was linear confirming that heat stress induced degradation could be
accounted for by thermodynamic effects of temperature. The cytosolic proteolysis of
HAHAB had anEa = 38.5±4.9 kcal/mole which was much larger than for cytosolic
proteolysis of TCA precipitable polypeptides but closely corresponded to the Ea of a
lysosomal mechanism reported above, as well as the lysosomal degradation of
endocytosed apolipoprotein B, Ea = 35 kcal/mole at 20-37°C (Bates, et al., 1982).
Together results suggested that “cytosolic” (3MA or chloroquine resistant) proteolysis
of aldolase B (HAHAB) was independent of autophagic sequestration (inhibition) and
acidic lysosomal pH but occurred by a complex mechanism with Ea similar to lysosomal
degradative mechanisms.
Lysosomal degradation of long-lived proteins is generally thought to be limited
by sequestration rate (high Ea), and sequestration can occur by non-autophagic (possibly
3 MA-resistant) process of receptor recognition and translocation (Dice and Chiang,
1988; Cuervo, et al., 1996). Chloroquine neutralizes the acidity of lysosomes and
inhibits lysosomal acid hydrolases, but proteolysis of HAHAB continued in the presence
of this drug. Consistent with this, aldolase A and B are thought to undergo limited
proteolysis by a neutral lysosomal peptidase (Horecker, et al. 1985). Though the data

123
are not conclusive, past investigators have shown that this peptidase might occur on the
cytosolic surface of lysosomes (Pontremoli, et al., 1982) which in itself could confer
resistance of the peptidase activity to 3MA and CHQ. Given these possibilities, 3MA or
CHQ resistant “cytosolic” proteolysis for aldolase B (HAHAB) in Figure 4-12 might
actually occur by lysosomal mechanisms. The highest Ea found published for cytosolic
proteolysis was Ea = 27 ± 5 kcal/mole reported for an enzyme now established as the
20S proteasome (Hough et al., 1986; Bond and Butler, 1987; Waxman, et al., 1987;
Coux, et al., 1996). Ea for HAHAB proteolysis was significantly greater than this (p <
0.019), suggesting a more complex mechanism like sequestration into lysosomes. The
mechanism of 3MA and chloroquine resistant proteolysis of aldolase B (HAHAB)
remains unidentified, but the results support that heat stress increases both lysosomal and
cytosolic mechanisms for the degradation of aldolase B by direct thermodynamic
stimulation of elevated temperatures.
A Model For the Degradation of Aldolase B
Figure 4-13 summarizes the results of the analysis above in a hypothetical model
for the degradation of aldolase B. It previously was established (Chapter 1) that
starvation for amino acids and serum (S) can induce autophagic degradation of aldolase
A and B (Fig. 4-13, Bl—»C—»D). In Chapter 3, evidence was provided that starvation
and heat stress (HS) induced ubiquitin-mediated autophagy (Fig. 4-13,
A—»B2—>C1—>C2—»D). Since it appeared that ubiquitinated aldolase B was enriched in
isolated autophagic vacuoles and lysosomes relative to cytosol, sequestration of aldolase

124
B appears to be enhanced by ubiquitination (B2>B1). Ubiquitination is known to
mediate cytosolic proteolysis of proteins into 6-9 amino acids long peptides (Coux, et al.,
1996; Lowe, et al., 1995) indicated by A—>E (lower arrow?)-»F (Fig. 4-13). Though
aldolase B occurs in ubiquitinated forms, when ubiquitination was inhibited (heat
stressed ts20 cells) an enhance cytosolic (3MA and CHQ resistant) proteolysis of
aldolase B still occurred (Fig. 4-13, A—»E (upper arrow)-»F).
The proteolysis at step F increased with temperature consistent with direct
thermal enhancement of chemical reactivity. The estimated activation energy (Ea = 35-
40 kcal/mole) was closer to a known value for a lysosomal pathway (Ea = 35 kcal/mole)
than for the major cytosolic protease, the 20S proteasome (Ea = 27 kcal/mole).
However, there was no difference from the 20S proteasome at a 0.01 significance level.
Furthermore, a basal level of ubiquitination can be measured in ts20 cells (Gropper, et
al., 1991), and ubiquitination is thought to be rate limiting (Ea > 27 kcal/mole) for
proteosomal degradation (Coux, et al., 1996), indicating that step F proteolysis might
still occur in the proteasome. Previously proposed mechanisms for limited proteolysis of
aldolase B on the cytosolic surface of lysosomes (Pontremoli, et al., 1982) and for non-
autophagic sequestration into lysosomes (Dice and Chiang, 1988) argue that the high Ea
of step F proteolysis might still involve lysosomes. Current data is insufficient to
definitively distinguish these possibilities. Here, cytosolic proteolysis was preferred as
the most likely possibility.
At higher temperatures, cytosolic proteolysis of aldolase B (Fig. 4-13, step F)
becomes equal to or greater than sequestration into lysosomes (step B). This

125
F. Proteolysis
CYTOSOL
B3
B. Sequestration
0.3%/h 130.5°C(CT)
{ 0.9%/h} |{37°C (TCA, S)}
1.7%/h
2.9%/h
39.5 C(HS)
37°C (S)
1.1%/h
1.9%/h
2.4%/h
30.5°c(CT)
37°C (ForS)
39.5°c(HS)
G. Degradation
1.5%/hJ (all)
amino acids
Ub-AldB*
Cl
I
LYSOSOME
AldB*
C. Proteolysis
C2
peptides
D2
AldB
D. Degradation
D1 'A
——►ammo acids
f
Figure 4-13: Pathways for Degradation of Aldolase B. Large stippled box, lysosomal
compartments (autophagic vacuoles and lysosomes); small box, aldolase B polypeptide
maintaining predominately native (solid outline) or denatured (broken outline)
conformation; AldB*, full length active enzyme; AldB, proteolytically processed inactive
enzyme; Ub-AldB, ubiquitinated aldolase B; peptides, hypothetical fragments of aldolase
B; arrows and upper case bold face letters, processes discussed in text; %/h, median
fractional rate of flux (SEM < 1/6 of magnitude shown, n = 24-43) through the
indicated process at temperature and condition: CT, control temperature; HS, heat
stress; F, fed; S, starved; “Proteolysis” indicates cleavage in to polypeptides;
“Degradation” indicates complete proteolysis to amino acids (TCA soluble). {37°C
(TCA S)}, rate of autophagic sequestration at 37°C for total TCA precipitable
polypeptides during starvation.

126
interpretation was made because during heat stress aldolase B degradation was
independent of autophagic degradation in lysosomes. Since TCA precipitable
polypeptides were still reliant on autophagic degradation, step F was considered to
produce peptides, consistent with involvement of the proteasome. Complete cytosolic
proteolysis to amino acids occurred (Fig. 4-13, step G) which was fairly constant with a
low temperature dependency, Ea = 5.1 kcal /mole, that was temperature-independent at a
0.01 significance level (Fig. 4-12, Cyto TCA). The balance of peptides produced in step
F are sequestered (step B3) and degraded (step D2) in lysosomes. According to TCA-
precipitable polypeptides, the autophagic degradation rate during starvation (TCA, S)
was 0.9%/h consistent with the temperature-dependency shown in Figure 4-12 (Lyso
TCA). However, in the same cells (Fao) under the same treatment (starvation),
lysosomal degradation of aldolase B was threefold higher, 2.9%/h (Fig. 4-13, step B,
(S)). This indicates that lysosomal mechanisms have a preference for degrading aldolase
B over TCA precipitated total proteins, suggesting an additional mechanism. In Chapter
5, a role for receptor-mediated targeting to lysosomes is demonstrated for aldolase B

CHAPTER 5:
SIGNAL-MEDIATED DEGRADATION OF ALDOLASE B
Introduction
Chapter 1 introduced the hypothesis that stress-induced degradation of aldolase
B requires ubiquitination and receptor-mediated targeting (Fig. 1-4). In Chapter 3,
evidence was presented that aldolase B was ubiquitinated and enriched in lysosomes for
a ubiquitin-mediated autophagic degradation during starvation and heat stress. In
Chapter 4, temperature increase caused an increase in cytosolic and lysosomal
degradation consistent with direct thermal stimulation of chemical reactivities. During
starvation for amino acids and serum, autophagic degradation of total TCA precipitated
polypeptides was consistent with the temperature-dependent rate predicted by thermal
stimulation at 37°C (Fig. 4-13, step B).
Lysosomal degradation of aldolase B in Fao cells was threefold higher than for
TCA precipitated polypeptides (Fig. 5-1), suggesting a mechanism preferential for
aldolase B sequestration into lysosomes. In Figure 5-1, the total height of each bar
indicates the total degradation rate during starvation for the indicated proteins, including
degradation rates for total long-lived proteins (first bar, Total), permanently expressed
recombinant aldolase B (third bar, HAHAB), and rate of loss for endogenous aldolase B
estimated on western blots (second bar, aldolase B). Each bar is divided into two parts.
127

128
7%
0%
TCA- Immuno- Immuno-
precipitated blotted precipitated
Total Aldolase B HAHAB
Protein Assayed
Figure 5-1: Starvation-Induced Lysosomal Degradation is faster for Aldolase B than for
Other Long-lived Proteins. Starvation-induced degradation was measured at 37°C as
previously described (Figs. 4-6, 4-9, and 4-10). Degradation of protein which was
resistant to lysosomal inhibition is superimposed on the total starvation-induced rate to
show the relative contribution of degradation (mean ± SEM, n = 8-24) occurring in
cytosol (cytosolic) and lysosomes (lysosomal). All cytosolic degradation rates were
comparable to each other; lysosomal degradation for Aldolase B or HAHAB was greater
than TCA-precipitated (Student’s t-test, p < 0.001).
The lower part indicates the degradation rate contributed by proteolysis in the cytosol
(Fig. 5-1, cytosolic, light-shaded part). These rates were calculated by inhibiting
lysosomes during starvation. They agreed with each other and were not statistically
different from basal degradation under fed conditions (Fig. 4-9). The results suggest that
cytosolic degradation of aldolase B probably follows a common basal mechanism used

129
by most long-lived proteins, consistent with the major role proposed for the cytosolic
proteasome (Coux, et al., 1996). Further, this indicated that in the presence of amino
acids (fed conditions) basal lysosomal degradation was too low to detect in Fao
hepatoma cells, consistent with the ability of liver-derived cells to respond to amino acid
concentrations by down-regulating protein degradation (Hendil, et al., 1990).
In Figure 5-1, the upper part of each bar represents starvation-induced
degradation that occurred in lysosomes (lysosomal, dark-shaded part) calculated by the
degradative rate lost by treatment with lysosomal inhibitors. Lysosomal degradation
accounted for the increase in protein degradation caused by starvation. During
starvation, lysosomal degradation of total long-lived proteins (TCA-precipitated) was
about 0.9%/hour, but lysosomal degradation of immunodetected aldolase B was much
higher at 2.9%/h. This indicated that aldolase B utilized an additional lysosomal
mechanism (relative to most long-lived proteins) that mediated ~2%/h greater rate of
proteolysis.
Intralysosomal degradation is rapid and limited by the rates of delivery of
proteins into lysosomes. Starvation can enhance two mechanisms for delivery of
cytosolic proteins into lysosomes: bulk uptake by autophagy and selective uptake by a
receptor-mediated mechanism. Above and in previous chapters, autophagic mechanisms
were considered. In this chapter, evidence demonstrates the existence of a lysosomal
targeting signal in aldolase B that specifically mediates starvation-induced degradation.
Lysosomal targeting signals have been characterized for other cytosolic proteins that

130
follow receptor-mediated delivery to lysosomes. A description of the proposed
mechanism for receptor-mediated targeting is presented in Chapter 1. Aldolase B had
characteristics of substrate proteins for this degradative mechanism, including three
potential targeting signal motifs (Fig. 1-2). These targeting motifs were mutated, and
starvation induced degradation of mutant protein was compared to wildtype. Below,
glutamine #111 is shown to be essential for the starvation-induced degradation of
aldolase B.
Transient Expression of RABM Mutations in Putative Lvsosome Targeting Signals
In order to examine a role for three potential lysosomal targeting signals in the
aldolase B sequence (Fig. 1-2), plasmid vectors for expressing RABM (rat aldolase B
with a carboxyl terminal myc epitope tag) were altered by site-directed mutagenesis to
change an “essential” glutamine (Q) in each signal to either threonine (T) or asparagine
(N) (Table 5-1). The three “essential” glutamines occurred at defined amino acid
positions in the aldolase B sequence: residue numbers 12, 58, and 111 (start methionine
= 1). A three letter “mutation code” was adopted here to label the data such that the
amino acid at positions 12, 58, and 111 were represented in order by the single letter
amino acid convention. In this manner, QQQ indicated wildtype, and QQN indicated
that Q111 was changed to asparagine (N).
At least seven site-directed mutant RABM’s representing all mutant permutations
needed to be expressed in cells at sufficient levels for biochemical detection. Given the
same efficiency as for the isolation of the FaoAB cell line (one permanent expressing line
per 340 G418-resistant Fao clones), 2,000-3,000 transfected lines would need isolated,

131
Table 5-1: Site-Directed Mutations in Aldolase B Sequence for Potential
Signal Motifs of Receptor-Mediated Targeting to Lysosomes
Labels Used
Amino Acid Residue** at the Essei
Position
for Each of Three Potentia
itial Glutamine
Motifs
Protein
Studied*
Mutation
Code
Site 1
#12
Site 2
#58
Site 3
#111
HAHAB
QQQ
(wt)
Q
Q
Q
RAB or Fao
endogenous
QQQ
(wt)
Q
Q
Q
RABM or
RABMQQQ
QQQ
(wt)
Q
Q
Q
RABMTQQ
TQQ
T
Q
Q
RABMQTQ
QTQ
Q
T
Q
RABMQNQ
QNQ
Q
N
Q
RABMQQT
QQT
Q
Q
T
RABMQQN
QQN
Q
Q
N
RABMTTQ
TTQ
T
T
Q
RABMTQT
TQT
T
Q
T
RABMQTT
QTT
Q
T
T
RABMTTT
TTT
T
T
T
*RAB, rat aldolase B; HAB, human aldolase B (97% identical to RAB; Fig. 1-2);
HAHAB,
HAB withl2CA5 epitope at amino terminus; RABM, RAB with 9E10 epitope at C-
terminus
** Single letter convention
amplified, and screened for permanent expression. To avoid this, other candidate cell
lines were screened for transfection efficiency (Table 5-2). Transfection efficiency was
defined as the fraction of cells expressing RABM by immunofluorescence screening.
Some commercial transfection lipid formulations were also screened and found that

132
transfection lipids operated differentially between cell lines. For example, Pfx-5
appeared to effectively transfect BHK cells but in other cell lines was toxic, produced an
inclusion body artifact, or failed to express RABM (Table 5-2).
The HuH7 human hepatoma cell line transfected with Pfx-3 (Invitrogen) had the
highest relative transfection efficiency for expressing RABM (Table 5-2), so isolation of
HuH7 cell lines permanently expressing RABM was attempted. Hundreds of HuH7
colonies isolated after transient transfection acquired resistance to the selectable marker
(G418), but none of forty clonal cell lines retained sufficient expression of recombinant
aldolase B for biochemical studies. This was consistent with the low incidence of
permanent expression of recombinant aldolase B in Fao cells, indicating a need for
massive screenings. However, the transient
Table 5-2: Optimization of Cell Line and Lipid Type
for Transient Expression of RABM*
Cell Lines
LIPID
Fao
HuH7
NRK
BHK
Pfx-1
toxic
toxic
0%
0.4-0.5%
Pfx-2
toxic
toxic
0%
1-2%
Pfx-3
toxic
2-3%
<0.05%
0%
Pfx-4
toxic
toxic
0%
0.3-0.4%
Pfx-5
toxic
inclusions
0%
0.2-0.3%
Pfx-6
toxic
toxic
<0.05%
1-2%
Pfx-7
0%?
n/d
<0.05%
0.5-1%
Pfx-8
inclusions
inclusions
inclusions
0.5-1%
DOTAP
tpxic
1-2%
<0.05%
0.3-0.4%
*%, fraction of cells immunofluoresently labeled for RABM-specific expression;
toxic, >50% cells had wrinkled appearance, obvious sloughing off present;
inclusions, large fluorescent inclusion bodies with little or no cytosolic labeling.

133
expression of RABM in HuH7 cells was relatively high and after optimization appeared
sufficient for biochemical assays (Table 5-3). Commonly, protocols call for plating cells
the day before transfection, but isolated individual HuH7 cells in cultures one day after
plating were killed by Pfx-3 treatment (data not shown) which limited transfection
efficiency (Table 5-3). By incubating cells for two days after plating, most cells
contacted other cells, survived Pfx-3 mediated transfection, and transfection efficiency
was increased by tenfold (Table 5-3). Though not generally examined during
optimization of transfection, the incubation time between plating cells and transfection
was found to have a large effect on transfection efficiency. Consistent transient
transfection efficiency of >15% (Table 5-3) indicated that transient transfection could
replace the more laborious isolation of permanent cell lines.
Table 5-3: Effects of Timing and Lipid:DNA Ratio on
Transfection Efficiency for RABM Expression in HuH7 Cell Line
Time After Plating
Transfection Time
Pfx-3 LipidrDNA Ratio
(DNA held constant)
days
hours
3:1
6:1
9:1
1
4
n/d
2%
n/d
2
4
20%
21%
21%
2
8
27%
22%
23%
In order to compare degradation rates for wildtype and mutant RABM,
expression levels for the mutants had to be sufficient. HuH7 cell transfection efficiencies

Figure 5-2: Transient Expression of Recombinant Aldolase B in HuH7 Cells. HuH7
human hepatoma cells were transfected for optimum transient expression of various
epitope-tagged aldolase B proteins defined in Table 4-1, processed for epitope-tag
specific immunofluorescence microscopy (9E10 for all, except 12CA5 for panel b), and
micrographs exposed until background labeling just became apparent; panels a through e,
representative fields showing labeling patterns for recombinant aldolase B proteins
defined in Table 5-1, similar fields occurred with expression of all the different proteins
except RABM’s with threonine (T) at position #58; panel f, expression of QTQ mutant
RABM showing the brightest cell on an entire coverslip, only rare cells were clearly
brighter than background but dim out-lining of nuclei was visible in many cells that did
not occur on untransfected control coverslips (not shown). Scale bar = 50 pM.

RABMQQT
óiówava
RABMTQQ
ÓMÓPMaV^
RABMQQQ
aVHVH
Lk>

136
were consistently greater than 15% for RABM proteins (Fig. 5-2, panels a-f) using
wildtype (QQQ) and site-directed mutants with changes in amino acid residues #12 or
#111 (TQQ, QQT, QQN, and TQT). Such high levels of expression were sufficient for
isolation by immunoprecipitation which confirmed comparable expression levels between
wildtype RABM and these mutants (Fig. 5-3). Note, the transiently expressed proteins
were similar for different mutants and wildtype RABM (41.3 kD) and did not occur in
cells transiently expressing (3-galactosidase ((3Gal). Previously during permanent
expression, RABM became associated with endogenous 40 kD aldolase which could be
co-immunoprecipitated (Fig. 4-8b). However, immunoprecipitation from transiently
transfected cultures pelleted very little or no 40 kD protein, indicating that transient
protein expression per cell was so high relative to endogenous aldolase expression that
the exogenous RABM predominately associated with itself presumably forming homo-
oligomeric complexes of RABM. Since transient overexpression excluded association
with endogenous aldolase, possible complications from oligomeric interactions with
endogenous aldolase subunits were reduced. For example, if all subunits of an aldolase
tetramer are degraded together, then wild type signals in an endogenous subunit could
rescue an associated mutant subunit. Transient expression seems to eliminate this
possibility. The results indicated that wildtype RABM and mutants at residues #12 and
#111 were expressed sufficiently for protein degradation measurements.
Replacing glutamine #58 with threonine (QTQ, TTQ, QTT, or TTT) prevented
high RABM expression (Fig. 5-2, panel f), and further biochemical analysis could not be

137
44.5
41.3
40.0
9E10
R «
O o
O GL
Aldolase B
J¡¡|k JBL
a
a
z
H
H
a
a
a
a
a
o
a
a
ca
H
a
a
CQ.
H
WBm
mm nv
42
41.3
Figure 5-3: Immunoprecipitation of RABM and its Mutants Transiently Expressed in
HuH7 Cells. HuH7 cell cultures were transfected for transient expression of p-
galactosidase (PGal) or RABM forms identified by the mutation codes shown in Table 5-
1; cells were then metabolically labeled with 35S-methionine then processed for anti-myc
epitope (left panel, 9E10) or for anti native aldolase B (right panel, Aldolase B)
immunoprecipitation as described in Materials and Methods; autoradiographs of dried
gels of immunoprecipitation pellets run on SDS-PAGE are shown; left and right panels
are lined up on a 44.5 kD heavy background protein (actin?), and differences in
electrophoretic migration between gels are indicated by relative positions of the 41.3 kD
band relative to the 44.5 kD band.

138
done (summarized later in Table 5-4). The minimal change in the cDNA (2 base pairs) is
unlikely to affect transfection efficiency. Consistent with this, many very low expressing
cells were apparent by very dim but visible negative nuclei suggesting that transfection
occurred, but the protein was not expressed well. To get around this low expression, a
less harsh change of glutamine #58 to asparagine (N) was tried which successfully
produced enough protein for degradation assays (Figs. 5-2, panel d, QNQ). In the QNQ
mutant, an amide side chain chemistry was retained at residue #58, suggesting a possible
role in aldolase B protein stability.
Other evidence supports a role for glutamine #58 in the structural stability of
aldolase B. First, glutamine #58 is embedded in the tertiary structure of the protein,
placing it in the right place for such a role. Glutamine #58 is not a highly conserved
residue which argues against this role. However, neighboring amino acid sequence was
examined in different mammalian aldolase isoforms and found that when glutamine #58
was missing then glutamine occurred at a position three residues away. These glutamine
side chains would occur on the same side of an a-helix in a similar 3-D position and
could perform the same structural role. Supporting this idea, glutamine #58 occurs in an
a-helical region of aldolase structure.
The results here provided wildtype and mutant RABM proteins transiently
expressed in HuH7 cells at similar levels (Figs. 5-2 and 5-3), allowing effects on
starvation induced degradation to be examined for mutations at all three potential signal
sequence motifs found in aldolase B

Starvation Induces Autophagic Degradation in HuH7 Cells
139
Before examining transiently expressed aldolase B (RABM), starvation-induced
degradation of total long-lived proteins in HuH7 cells was examined. Chloroquine
(CHQ) but not 3-methyladenine (3MA) significantly reduced basal degradation of long-
lived proteins in HuH7 cells (Fig. 5-4). This suggested that a fraction of unstressed basal
protein degradation occurred in lysosomes but not by 3MA-sensitive autophagy. Both
starvation-induced
Inhibitor Treatment
Figure 5-4: Starvation-Induced Autophagic Degradation of Long-lived Proteins Occurs
in HuH7 Cells. HuH7 cultures were metabolically labeled with 14C-valine, chased with
10 mM “cold” valine in DMEM+10% FBS (Fed) or Krebs-Heinseleit without amino
acids (starved), and loss of TCA precipitable counts was used to calculate degradation
rates (mean ± SEM, n = 12) as described in Materials and Methods; media were
unsupplemented (control) or supplemented with indicated concentrations of chloroquine
(CHQ) or 3-methylyadenine (3MA); Student’s t-tests: inhibitor treated < corresponding
control degradation rate (*, p < 0.013); Starved > Fed degradation rate (**, p < 0.01;
***, p < 0.005, ****, p < 0.0001).

140
(Starved) and basal (Fed) degradation of long-lived proteins was inhibited with
chloroquine (CHQ) such that starvation appeared to induce a mechanism that was
resistant to CHQ (Fig. 5-4). Alternatively, the results were consistent with mechanisms
for fed and starved degradation having a similar sensitivity to CHQ. Generally,
lysosomal degradation plays a greater role in starvation-induced degradation (Starved)
than in basal degradation (fed). Since the results were inconsistent with this, HuH7 cells
might have a non-specific sensitivity to CHQ which affects both basal and starvation-
induced degradation equally. Doubling inhibitor concentration did not change results
(Fig. 5-4), indicating that a maximal effect occurred, but the possibility that lower CHQ
concentrations might be more specific for starvation-induced degradation in HuH7 cells
was not examined. 3-methyladenine specifically reduced the enhanced degradation of
total long-lived proteins by about 0.8%/h in HuH7 cells (Fig. 5-2), consistent with the
0.9%/h inhibition of enhanced autophagic degradation in Fao cells (Fig. 4-6). The
results suggested that starvation-induced autophagic degradation of total long-lived
proteins occurred in HuH7 cells.
Transient Expression Does Not Affect Starvation-Induced Degradation of RABM
I showed that HuH7 cells undergo starvation-induced degradation of long-lived
proteins and that 9E10 myc-epitope tagged rat aldolase B (RABM) could be transiently
expressed at sufficient levels for biochemical analysis. In this section, the degradation of
the transiently expressed RABM is shown to be comparable to permanently expressed
recombinant aldolase B (HAHAB) and endogenous aldolase B of Fao cells.

141
I found that variation in permanent expression levels had little or no effect on
degradation rates in E36 cells (data not shown), but a sub-population of the transiently
transfected HuH7 cells clearly had higher RABM levels per cell than previously
examined. Thus, it was important to establish whether transient transfection altered the
degradation of
£
OQ
(A
TO
o
<
M-
o
c
o
U-*
re
-a
2
o
â–¡
RAB HAHAB RABM HAHAB
(endogenous) (permanent) (transient) (transient)
Fao FaoAB HuH7 HuH7
Aldolase Form (expression) & Cell Line
Figure 5-5: Differences in Expression and Epitope Tag Have No Effect on Starvation-
Induced Degradation of Aldolase B in Hepatoma Cell Lines: Cultures of cell lines
indicated expressing aldolase B were measured for fed and starved aldolase B
degradation rates ± standard error (n = 25 to 37) according to protocols compatible for
each form of the enzyme as described in Materials and Methods. RAB (endogenous)
Fao, endogenous rat aldolase B in Fao rat hepatoma cells; HAHAB (permanent) Fao,
permanently expressed amino HA-tagged human aldolase B transfected in Fao cells;
RABM (transient) HuH7, transiently expressed carboxyl Myc-tagged rat aldolase B in
HuH7 cells; HAHAB (transient) HuH7, transiently expressed HAHAB in HuH7 human
hepatoma cells. Student’s t-test: Starved > Fed (*, p < 0.0005); HAHAB (Starved) in
HuH7 > in FaoAB and RAB (starved) in Fao > RABM in HuH7 (**, p < 0.001)

142
aldolase B. Figure 5-5 compares the basal and starvation-induced degradation of various
forms of aldolase B expressed in various ways. The results are similar regardless of the
variations. There was little or no effect caused by epitope tagging. Tagging at the
amino terminus or the carboxyl terminus gave identical results, and using 12CA5 versus
9E10 epitope also had no effect. Whether endogenously expressed, permanently
expressed from transfected cDNA, or transiently expressed as described above the
results were similar (Fig. 5-5).
Site-Directed Mutations Did Not Affect Wildtvpe Activity of RABM
Endogenous 40 kD aldolase was absent in proteins immunoprecipitated from
transiently transfected cells (Fig 5-3), indicating that characteristics of
immunoprecipitated RABM could be examined in isolation from endogenous aldolase
effects. Antiserum against aldolase B also failed to pull down endogenous aldolase
indicating that the endogenous isoform of HuH7 cells is probably aldolase A (Fig. 5-3).
These results indicated that exogenous RABM is for the most part separate from
endogenous aldolase, during transient expression.
Even though endogenous aldolase was not associated with transiently
overexpressed RABM, a 42 kD protein was specifically immunoprecipitated from
RABM expressing but not p-galactosidase expressing cells (Fig. 5-3). This indicated
that RABM associated with an endogenous 42 kD protein. Aldolase isozymes have an
established binding affinity for

143
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has a molecular weight of
42 lcD. The results suggest that RABM retains GAPDH binding activity. If so, then all
the mutants examined had no effect on GAPDH binding, because the 42 kD protein was
co-immunoprecipitated with them as well (Fig 5-3).
With transient expression, immunoprecipitation was used to isolate epitope-
tagged aldolase B away from endogenous aldolase, giving purified RABM or HAHAB.
By measuring activity specifically precipitated by the epitope tag, the effect of site-
directed mutations on the enzymatic activity of aldolase B could determine. As shown in
Figure 5-6, all forms of recombinant aldolase B tested had similar activity regardless of
epitope tag or site-directed mutations. Thus, effects of mutations on starvation-induced
degradation shown below are not due to indirect effects of altered aldolase B activity
(Fig. 5-6).
Glutamine Residue #111 is Required for Starvation-Induced Degradation of Aldolase B
Above, the degradation of RABM transiently expressed in HuH7 cells was shown
comparable to the degradation of permanently expressed HAHAB and endogenous RAB
(Fig. 5-5). These aldolase B forms followed a starvation inducible pathway for
degradation. Then mutant forms of RABM were transiently expressed in HuH7 cells;
the different mutations are described and defined in Table 5-1.

144
RABM RABM RABM RABM RABM RABM beta-Gal HAHAB beta-Gal
QQQ TQQ QNQ QQN QQT TQT (9E10) QQQ (12CA5)
123456789
Protein Expressed
Figure 5-6: Different Forms of Recombinant Aldolase B Retain Similar Levels of
Catalytic Activity. Replicate HuH7 cell cultures were transfected for transient
expression of indicated proteins, lysed in 1% NP40/1 mM EDTA/TBS pH 7.5, and
immunoprecipitated for 9E10 (bars 1 through 7) or 12CA5 (bars 8 and 9) epitopes as
detailed in Materials and Methods under subsaturating conditions. After standard
washes, pellets were washed with TE pH 7.5 then suspended in 1 ml aldolase reaction
mixture. Periodically, each sample was pelleted and OD(340 nm) of supernatant was
measured. The mean ± SEM (n = 3) OD decrease caused by adding FDP was
normalized to the non-specific background of cultures expressing P-galactosidase and
immunoprecipitated with the same antibody (bars 7 or 9). Student’s t-test: relative
activity for RABM or HAHAB expressing cultures > untransfected non-specific
controls, lanes 7 and 9 (p < 0.0001).
Figure 5-7 shows that glutamine #111 is essential for starvation-induced
degradation of RABM. Changing at glutamine #12 to threonine (TQQ) yielded fed and
starved degradation rates for RABM that were statistically indistinguishable from
wildtype aldolase B sequence (QQQ). Even though the peptide sequence at glutamine
#12 is the most conserved of the mutated signals (identical in all vertebrate aldolases), it

145
did not function as a signal for starvation-induced degradation (Fig. 5-7). A role for
glutamine #58 could not be assessed by threonine substitution, because this resulted in
expression too low for degradation assays. When glutamine was replaced with
asparagine at residue #58 (QNQ), conserving an amide at this residue, expression was
close to other high level transient expressions, and starvation-induced degradation was
like wildtype (Fig. 5-7). The results indicated that glutamines at residues #12 and #58
were not essential for starvation-induced degradation and suggested that the lysosomal
targeting motifs at these positions were not functional in vivo.
Whenever the glutamine at residue #111 was mutated then starvation-induced
degradation of RABM was completely inhibited (Fig. 5-7). For wildtype aldolase B
sequence in RABM (QQQ), starvation increased degradation of RABMQQQ by greater
than twofold, but when glutamine residue #111 was changed to asparagine (QQN) or
threonine (QQT and TQT), the resulting mutant RABM failed to show an increase in its
degradation in response to starvation. Since the loss of inducible degradation occurred
even with a minimum change of glutamine to asparagine (QQN), glutamine #111 is
clearly essential for starvation-induced degradation of aldolase B and asparagine cannot
function in its place. This is consistent with the function of a motif originally described
by J. Fred Dice and indicates that aldolase B is a substrate for receptor-mediated
targeting to lysosomes.

146
RABM Form
Figure 5-7: Glutamine Residue #111 is Required for Starvation-Induced Degradation of
Aldolase B. Various forms of RABM were transiently expressed in HuH7 cells, then fed
and starved degradation rates ± standard error (n = 18 to 33) for RABM were measured
as described in Materials and Methods. Different forms of RABM are labeled according
to the mutation code given in Table 5-1. Student’s t-test: *, Starved > Fed, p<0.0001;
**, mutant < wildtype = QQQ, p < 0.0001.
Glutamine #111 Specifically Mediates Starvation-Induced Degradation
Above, it was demonstrated that changing glutamine #111 of aldolase B affects
its starvation-induced degradation. Here, it is proposed that this change directly affects a
degradative mechanism, but if some other function of this enzyme is blocked, then
inhibition of starvation-induced degradation might be an indirect affect of other lost
function. To address this, evidence that various aldolase functions were similar between

147
the RABM containing wildtype sequence (QQQ) and mutants at glutamine #111 (QQN,
QQT, and TQT) was collected.
Table 5-4 summarizes evidence that mutations at glutamine #111 were specific
for blocking the starvation-induced degradation of aldolase B. First, expression levels
were similar to wild type RABM, including transfection efficiencies (Table 5-4, column
2) and immunoprecipitated RABM (Fig. 5-3). Second, aldolase is known to bind f-actin
(O’Reilly and Clarke, 1993; Wang, et al., 1996), and HuH7 cells have well developed
stress fibers that became decorated with antibodies specific for RABM (Fig. 5-8, 9E10).
Localization of RABM to actin was confirmed by double labeling with phalloidin which
specifically labels f-actin (Figure 5-8b). For all mutant RABM’s used in degradation
experiments, similar stress fiber morphology (Stress Fiber Label) was observed as with
RABM-specific label in RABMQQQ wildtype expressing cells (data not shown). This
provided evidence that some actin-binding activity was retained in these mutants (Table
5-4, column 3) Third, aldolase is known to bind GAPDH (Clarke et al., 1982), and
when mild conditions were used for immunoprecipitations (Fig. 5-3, Aldolase B) a 42
kD protein was specifically pulled down. Though its identity was not confirmed, the
sized matched that predicted for the 42 kD subunits of GAPDH (Clarke et al., 1982).
The results suggest that GAPDH binding was retained by site-directed mutants of
RABM (Table 5-4, column 4). Above, enzymatic activities were already shown to be
comparable to each other regardless of the mutations (Fig. 5-6, Table 5-4, column 5).
Lastly, there were no significant differences between various basal degradation rates for
all the assayed RABM forms, including the mutants. Furthermore, mutation of

148
Table 5-4: Summary of RABM Forms Expressed in HuH7 Cells
RABM
Form1
Transient
Expression
(% Brightly
Labeled Cells)
Stress
Fiber
Label2
Binds to
a 42 kD
protein3
Aldolase
Activity4
Basal
Degradation
of RABM5
Starvation-
Induced
Degradation
of RABM6
000
10-25%
+
+
+
+
+
TOO
10-25%
+
+
+
+
+
QNQ
5-15%
+
+
+
+
+
QQN
10-25%
+
+
+
+
—
QQT
10-25%
+
+
+
+
—
tqt
10-25%
+
+
+
+
—
Mutants Expressed too Low for Additional Characterization
rn/d, not done):
QTQ
<0.1%
±
n/d
n/d
n/d
n/d
TTQ
<0.1%
±
n/d
n/d
n/d
n/d
QTT
<0.1%
±
n/d
n/d
n/d
n/d
QTN
<0.1%
±
n/d
n/d
n/d
n/d
TTT
<0.1%
±
n/d
n/d
n/d
n/d
1 See mutation codes in Table ¿
-2
2 + : common, often bright; ± : occasional, usually dim, observation based on rare labeling
3 + : specific co-precipitation of an endogenous 42 kD protein (probably GAPDH), see Fig. 4-28
4 + : activity in immunoprecipitate lacking 40 kD protein (endogenous aldolase), see Fig. 4-29
5 + : basal degradation = published values (1 0-2.5%/h); see Fig. 4-31
6 + : starved degradation > basal; - : starved degradation = basal; see Fig. 4-31
glutamine #111 specifically inhibited the induced portion of RABM degradation,
indicating that the basal degradation of mutated RABM’s continued during starvation.
The data support that the basal degradation of RABM continued without being affected
by the site-directed mutations (Table 5-4, column 6). Together the results demonstrate
that glutamine #111 in aldolase B is likely to play a direct role in receptor-mediated
targeting to lysosomes induced by starvation.

149
a. BHK HuH7
Figure 5-12: RABM has Actin Binding Activity, a) left panel, BHK (baby hamster
kidney) and right panel, HuH7 cells were transfected for transient RABM expression and
single labeled for 9E10 immunofluorescence microscopy as described in Materials and
Methods, field showing example of occasional cells having good stress-fiber labeling
indicated by arrows; b) HuH7 cells were labeled as in (a) and then double stained with
phalloidin carrying an alternative fluorophore, the same field is shown: left panel, the
9E10 fluorescence channel, right panel, the phalloidin fluorescence channel.

150
At the end of Chapter 3 and the beginning of this chapter, starvation-induced
degradation of aldolase B was examined in hepatoma cell lines. It was confirmed that
starvation-induced degradation occurs by autophagic degradation for long-lived proteins
in general including aldolase B. I found a residue, glutamine #111 that was specifically
essential for starvation-induced degradation of aldolase B, indicating that it served as
part of a degradative targeting signal. Together, the results of Chapters 3 through 5
support the hypothesis that stress-induced degradation of aldolase B requires
ubiquitination and a receptor-mediated targeting signal.

CHAPTER 6:
SUMMARY AND CONCLUSIONS
Introduction
A model was presented showing two mechanisms known to mediate stress-
induced degradation of long-lived proteins (Fig. 1-4). In Figure 6-1, the findings of this
study were used to update the initial model with aldolase B utilized as a representative
long-lived cytosolic protein. Evidence indicated that autophagy, ubiquitination, cytosolic
proteolysis, and a molecular recognition signal function in the degradation of aldolase B
in response to heat stress and starvation.
Autophagy and Ubiquitination
This study began by identifying ubiquitinated forms of aldolase B with antibodies
raised against ubiquitin-free aldolase B. In Figure 6-1, ubiquitination is represented by
process 6 which maintains aldolase B partially denatured as indicated by preferential
reactivity with antibodies to denatured epitopes (Fig. 3-2). Unmodified aldolase B
predominated in cytosol, whereas ubiquitinated aldolase B was enriched in lysosomal
compartments, especially autophagic vacuoles (Fig. 1-3 and 3-3). Furthermore,
lysosomal association of a major ubiquitinated form of aldolase B (Ub68) increased
during amino acid starvation and decreased when autophagy was inhibited. Ub68 also
occurred in E36 cells that undergo increased autophagic degradation during heat stress
151

152
Figure 6-1: Mechanisms for Stress-Induced Degradation of Cytosolic Proteins in
Lvsosomes. Autophagy (upper pathway) and receptor-mediated targeting (lower
pathway) were proposed for stress-induced delivery of cytosolic proteins to lysosomes
for degradation; the arbitrary cytosolic protein is shown as a tetramer (aldolase B occurs
as a tetramer); components of the pathways are labeled on the diagram; processes are
labeled by boxed numbers: la & b, association with or engulfment by pre-autophagic
membranes; 2, sequestration into double-membrane bound autophagic vacuole; 3,
maturation of autophagic vacuole (acidification and acquisition of lysosomal hydrolases);
4a & b, proteolysis into polypeptide fragments; 5, complete degradation to amino acids;
6, disassembly and denaturation of structure by an unknown factor; 7, association with a
receptor complex on the lysosomal surface; 8, translocation across the lysosomal
membrane; 9, limited proteolysis and removal of ubiquitin; 10, proteolysis into
polypeptide fragments; 11, complete degradation to amino acids.
(Fig. 3-11). In response to heat stress, endogenous aldolase A of E36 cells accumulated
in lysosomes dependent upon elevated levels of ubiquitination (Fig. 3-5). These results

153
suggested that ubiquitination facilitated the autophagic delivery of aldolase B to
lysosomes (Fig. 6-1, process lb > la), but other data indicate that autophagic
degradation is affected at a later step in the pathway.
Ubiquitination was previously proposed to mediate degradation in
autolysosomes (Fig. 6-1, process 4) rather than sequestration, because autolysosomes
expanded sixfold in volume when ubiquitination was inhibited (Lenk et al., 1992).
Consistent with this, lysosomal degradation was temperature-dependent (Fig. 6-12),
increased approximately 6-fold during heat stress relative to control temperature (Fig. 6-
13), and required ubiquitination (Figs. 3-14 and 3-15). Additionally, ubiquitination was
required for limited proteolytic processing of an epitope-tagged form of aldolase B
associated with lysosomes (Fig. 3-10). This evidence supports a role for ubiquitination
in proteolysis mediated by at least some lysosomal proteases (Fig. 6-1, process 9).
Whether for delivery or degradation, this study demonstrates that ubiquitination
mediates lysosomal mechanisms for degradation of aldolase B. The hypothesis that
aldolase B requires ubiquitination for enhanced degradation in lysosomes was supported.
Given this, a number of questions arise. What must be ubiquitinated, aldolase B or a
component of the autophagic mechanism? Is ubiquitinated aldolase B sequestered or
degraded faster than unmodified aldolase B? What does ubiquitination do to the
structure and function of aldolase B?
Preliminary evidence indicates that ubiquitinated aldolase B is maintained in a
more denatured conformation. Does ubiquitination mediate disassembly of aldolase

154
tetramers? Does it expose potential targeting signals for degradative mechanisms? Is
ubiquitinated aldolase B preferentially degraded? Ubiquitinated aldolase B constitutes at
least part of Ub68, one the most abundant protein-ubiquitin conjugate species
characterized on SDS-PAGE. Though enriched in lysosomes relative to total proteins, a
significant amount of Ub68 still occurs in the cytosol (Fig. 1-3 and 3-3). Does aldolase
B make up most or all of Ub68? How abundant is Ub68 relative to unmodified aldolase
B? Is Ub68 a necessary intermediate for ubiquitin-mediated degradation of aldolase B?
Could ubiquitinated aldolase B perform another function?
Clues from Temperature-Dependent Cytosolic Proteolysis and Lysosomal Degradation
While examining heat stress-induced autophagic degradation, a second
mechanism for aldolase B degradation during heat stress was identified. The degradative
activity was resistant to treatments that inhibited autophagy and lysosomal degradation,
suggesting a cytosolic mechanism (Fig. 6-1, processes 10 and 11). Temperature-
dependency of this cytosolic mechanism was consistent with thermal enhancement of a
rate-limiting reaction. Calculations of the activation energy (Ea) were limited by use of
only three temperatures (Fig. 4-12), but the large magnitude was consistent with
regulated degradation by the complete 26S proteasome complex. An initial Ea estimate
was 28 kcal/mole (data not shown), but when more data were included Ea was 35-40
kcal/mole. This was higher than predicted for proteolysis by isolated 20S proteasomes
(27 kcal/mole), supporting the concept that a rate limiting step for proteasomes occurs
before 20S-mediated proteolysis by 19S regulatory components of the entire 26S

155
proteasome complex (Coux, et al., 1996). However, Ea’s estimated for cytosolic
proteolysis of aldolase B could not distinguish between Ea’s predicted for autophagic
degradation (35 kcal/mole) and regulated proteolysis in 26S cytosolic proteasomes
(presumably >27 kcal/mole). The inhibitors used here measurably blocked lysosomal
degradation of total TCA proteins, indicating that a cytosolic protease, consistent with
26S proteasomes, mediated proteolysis of aldolase B.
Stress-induced degradation in proteasomes is known to be regulated by
ubiquitination, but when ubiquitination was inhibited, cytosolic proteolysis of aldolase B
continued. However, inhibition of ubiquitination was incomplete (Gropper, et al., 1991)
and could not eliminate the possibility that cytosolic proteolysis continued at low levels
of ubiquitination. Consistent with this, similar amounts of Ub68 (ubiquitinated aldolase
B) occurred in cells having a predicted fourfold difference in ubiquitination activity (Fig.
3-11).
During heat stress, lysosomal inhibition did not affect enhanced cytosolic
proteolysis of aldolase B (Figs. 4-4 and 4-11) but effectively blocked enhanced
degradation of total long-lived TCA-precipitable peptides (Fig. 3-15). If aldolase B is
representative of long-lived housekeeping proteins, then enhanced cytosolic proteolysis
of aldolase B (loss of immunoreactivity) was limited to production of undetected TCA
precipitable polypeptides, and enhanced degradation of total proteins (loss of TCA
precipitability) proceeded to amino acids in lysosomes via autophagy. The results are
consistent with a role for proteasomes in aldolase B degradation, because cytosolic

156
proteasomes produce small polypeptides rather than amino acids (Lowe et al., 1995;
Coux, et al.,1996), whereas lysosomes completely degrade proteins to amino acids
(Hershko and Ciechanover, 1982; Mortimore and Poso, 1987; Olson et al., 1990).
Autophagic degradation of TCA precipitated proteins and partial cytosolic
proteolysis of aldolase B correlated with temperature such that their rates correlated with
each other (Fig. 4-12). This suggested that both mechanisms could play a role in heat
stress-induced degradation of aldolase B by thermodynamic stimulation of molecular
machinery. Cytosolic partial proteolysis was faster than autophagic degradation under
similar conditions (Fig. 4-13), suggesting the possibility that a product of the former
might stimulate the latter. Are lysosomes regulated by products of proteolysis? Amino
acids (lysosomal products) have been established to inhibit autophagic degradation. Is it
possible that peptides produced by cytosolic proteases like the proteasome have an
opposite effect, stimulating autophagy?
Endogenous inhibitors of the proteasome have been proposed to prevent
excessive proteolysis of cellular proteins (Coux, et al., 1996). One of the best
characterized endogenous inhibitors, CF-2, was found to be identical to 8-aminolevulinic
acid dehydratase (AADH), an essential enzyme in heme biogenesis (Guo, et al, 1994).
AADH is a long-lived oligomeric housekeeping protein with 40 kD subunits that occur
as a 50 kD ubiquitinated form associated with 26S proteasomes (Guo, et al, 1994).
Above, aldolase B was described as a long-lived oligomeric housekeeping protein with
40 kD subunits that occur as a 68 kD ubiquitinated form. The parallel suggests that

157
aldolase B might be another endogenous proteasome inhibitor. Consistent with this, the
proteasome has a stable intermediate during carboxyl to amino terminal proteolysis of
substrates (Lowe et al., 1995), and aldolase B has a protease-sensitive carboxyl terminus
(Chapter 1). The proteasome might begin proteolysis on the accessible carboxyl
terminus but be blocked by the protease-resistant structure for the rest of aldolase B.
Stress-induced ubiquitination of aldolase B could relax its structure, allow it to degrade,
and thereby make the proteasome available to degrade other proteins. Though the
identity of the cytosolic protease involved in the degradation of aldolase B still needs to
be established, a role for the proteasome seems important to examine.
Signal-Mediated Targeting
Aldolase was found to have properties similar to substrates of receptor-mediated
targeting to lysosomes (Fig. 6-1, processes 7 and 8), including three potential targeting
signals in the aldolase B sequence (Fig. 1-2). Site-directed mutagenesis was used to
replace glutamines defined as “essential” for each of these motifs. Of these, only
glutamine #111 was shown to specifically mediated starvation-induced degradation of
aldolase B. Whether aldolase B utilizes the same mechanism as previously described for
receptor-mediated targeting to lysosomes was not determined, but the data are
consistent with this mechanism. Previously, receptor function for this pathway was
demonstrated in cultured cells (Cuervo, et al., 1996), but signal function had not. If
glutamine #111 mediates this mechanism for aldolase B, then this study provides the first
evidence for signal function in living cells.

158
A major weakness of receptor-mediated targeting to lysosomes was recently
published (Gorinsky, et al., 1996). Conformations of known substrates for this pathway
would prevent receptor recognition, prompting investigators to propose unknown
factors to create more extended conformations (Gorinsky, et al., 1996). Based on
native and denatured immunoreactivities, ubiquitinated aldolase B retained a more
denatured conformation than unmodified aldolase B. This suggested that ubiquitin can
function as the unknown factor of Figure 1-1 to relax the structure of aldolase B and
improve lysosomal targeting signal availability (Fig. 6-1, process 6 and 7).
While characterizing the signal-mediated degradation of aldolase B, rat and
human aldolase B were expressed in rat and human cells. Maximal degradation rates
occurred when the source animal species of the aldolase B matched the species of the
cell (Fig. 5-5, RAB in Fao and HAHAB in HuH7). When species did not match
(HAHAB in Fao and RABM in HuH7), starvation-induced degradation rates were 35-
40% lower (Student’s t-test, p < 0.001). This indicated that species specific differences
between rat and human aldolase B might mediate recognition by proteolytic machinery.
Between rat and human aldolase B, there are seventeen non-identical amino acid
residues; only twelve alter side chain chemistry (Fig. 1-2). These residues are not near
the primary amino acid sequence for the signal at glutamine #111, indicating that some
might function as an independent recognition signal for cellular degradative mechanisms.
Though more work is needed to confirm these initial findings, the ability of cells to
distinguish between rat and human isoforms would further support signal-mediated

159
targeting of aldolase B during starvation. This study supports the hypothesis that a
molecular signal mediates stress-induced mechanisms for degradation of aldolase B.
Present and Future Contributions to the Field of Protein Turnover
This study demonstrates that Aldolase B is a substrate for multiubiquitination and
ubiquitin-mediated proteolysis in lysosomes. This is consistent with the hypothesis that
ubiquitination of substrate proteins facilitates degradation in lysosomes (Lenk, et al.,
1992). A secondary hypothesis is that ubiquitination is required for a subpopulation of
lysosomal hydrolases. In support of this, limited proteolysis of aldolase B in lysosomes
was blocked when ubiquitination was inhibited (Fig. 3-10), and aldolase B did not
significantly accumulate in lysosomes, indicating other proteolytic activities continued.
In the future, the recombinant aldolase B (RABM) can be used as a marker for ubiquitin-
dependent limited proteolysis in lysosomes. As such, limited proteolysis of RABM could
serve as an assay for this activity, allowing purification and identification of specific
lysosomal components that require ubiquitination.
When aldolase A was examined, heat stress cause greater than twofold
accumulation of aldolase enzyme activity in lysosomes, and this accumulation was
ubiquitin-dependent (Fig. 3-5). This indicated that ubiquitination plays a role in
sequestration of aldolase A. In support of this interpretation, ubiquitinated forms of
aldolase B (Ub68) were enriched more in autophagic vacuoles than in cytosol or
lysosomes (Fig. l-3a). Together, the data suggest that ubiquitination mediates at least
two processes during lysosomal degradation: sequestration and lysosomal proteolysis.

160
Autophagic sequestration in starved mammalian cells was proposed to occur by
bulk non-selective uptake (Kopitz et al., 1990). However, in starved liver cells,
ubiquitinated aldolase B was enriched in lysosomes, while unmodified aldolase B was
enriched in cytosol (Fig. 3-3). Assuming ubiquitination occurs in cytosol, the results
contradict a non-selective mechanism, and suggest that ubiquitinated proteins can
undergo selective sequestration. Selective autophagy occurs in yeast (Tuttle et al.,
1993). Aldolase B might provide valuable evidence for ubiquitin-mediated selective
autophagy. The different SDS-PAGE mobility of 40 kD unmodified and 68 kD
ubiquitinated (Ub68) aldolase B can be used to separate them after co-
immunoprecipitation (Fig. 3-11). In pulse-chase experiments, the relative loss of
radiolabel from these two forms could be compared with and without autophagic
inhibition. The results would determine relative degradation rates for aldolase B and
Ub68 and their relationship with autophagic mechanisms. If ubiquitination of aldolase B
makes it a better substrate for autophagy, then autophagic degradation of Ub68 would
be more rapid than for 40 kD aldolase B.
Amino acids have long been known to inhibit autophagic degradation, but how
their levels are detected and translated into an inhibitory signal is unknown.
Temperature-dependence for cytosolic proteolysis of aldolase B and lysosomal
degradation of long-lived proteins were parallel with statistically equal activation
energies (Fig. 4-12). Equal activation energies are predicted for processes controlled by
the same rate limiting mechanism. If cytosolic proteolysis of long-lived proteins is partial

161
as proposed for aldolase B above, then cytosolic peptides would be produced. If these
peptides induce autophagy, then autophagic degradation would be dependent on the
rate-limiting step for cytosolic proteolysis and give the same Ea. This suggests a model
in which amino acid deprivation induces limited proteolysis which produces peptides
which induce autophagy. Autophagic degradation in lysosomes produces amino acids
that might inhibit cytosolic proteases that produce peptides, causing peptide levels and
peptide-dependent autophagy to be reduced. Such a model could be tested by
introducing peptides into cytosol with liposomes, electroporation, microinjection or
overexpression from plasmid vectors then measuring autophagic degradation. If the
model is correct, then peptides should induce autophagy even in the presence of
inhibitory amino acids.
As discussed previously, the proteasome is the major protease in cytosol, is
known to produce peptides, and might mediate cytosolic proteolysis of aldolase B. To
examine this, chemical inhibitors of proteasomes can be tested for their ability to block
cytosolic proteolysis of aldolase B. This will have to be done in the presence of
lysosomal inhibition to detect the cytosolic protease activity while excluding lysosomal
degradative mechanisms. It was also proposed that aldolase B might be an endogenous
inhibitor of proteasomes. Proteasomes have been effectively purified from cells, and in
vitro inhibition of its activity aldolase B can be tested. This study purified catalytically
active recombinant aldolase B expressed in E. coli. The protease-sensitive carboxyl
terminus of aldolase B would be a reasonable site for initiation of proteolysis by the

162
proteasome. Carboxyl-terminally truncated recombinant aldolase B could be purified to
determine if the “loose” carboxyl terminus is necessary for interaction with the
proteasome. In this way, it might be shown that aldolase B is a new endogenous
inhibitor for the proteasome or a mechanism for protein interaction with the proteasome
might be established.
The last major find in this study is the existence of a targeting signal in aldolase B
required for starvation-induced degradation. Whether aldolase follows the currently
proposed mechanism for receptor-mediated pathway is not known. This could be
established by in vitro assays developed by J. Fred Dice using known substrates to
compete for the pathway. Transient overexpression of the receptor for this pathway,
LGP96, in cultured cells was shown to more than double long-lived protein degradation
(Cuervo et al., 1996). In this study, transiently expressed aldolase B had degradation
rates similar to those for endogenously expressed aldolase B. By coexpressing LGP96
and aldolase B, degradation of aldolase B should be increased, and this increase should
not occur for aldolase B with a mutated targeting signal (altered glutamine #111). This
would demonstrate that aldolase B is a bonafide substrate for the established pathway
and would be the first demonstration of signal function in living cells. If aldolase B
degradation is not mediated by LGP96, then an alternative recognition mechanism or
receptor would be indicated for aldolase B. If so, future effort would focus on
characterizing the nature of the signal in aldolase B and identifying the novel recognition
molecules mediating its starvation-induce degradation.

163
In conclusion, ubiquitinated forms of aldolase B (e.g. Ub68) associated with
autophagic vacuoles and lysosomes during nutrient stress (starvation) in rat liver and the
Fao rat hepatoma cell line. Ubiquitinated aldolase B that maintained a denatured
conformation possibly exposing recognition signals was detected in rat liver, Fao cells,
and in the E36 Chinese hamster lung cell line. During heat stress, accumulation of
endogenous aldolase A in lysosomes and lysosomal proteolytic processing of exogenous
aldolase B required ubiquitination in E36 cells. Heat stress caused ubiquitin-mediated
autophagic degradation of long-lived proteins in E36 cells. Together the results support
a role for ubiquitination in the stress-induced degradation of aldolase B. Basal and
starvation-induced degradation of transiently overexpressed aldolase B was like that for
endogenous aldolase B, in the HuH7 human hepatoma cell line. A mutated (one amino
acid changed) lysosomal targeting signal in aldolase B specifically prevented starvation-
induced degradation of the mutant protein with no effect on other aldolase B properties.
Together, these results supported the original hypothesis that during stress, aldolase B
requires both ubiquitination and a receptor-mediated targeting signal for enhanced
degradation in lysosomes.

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BIOGRAPHICAL SKETCH
In 1986, Peter P. Susan earned his Bachelor of Science degree from the
Pennsylvania State University at State College, Pennsylvania. Early in his studies he
majored in computer science then changed his major to microbiology. When finishing his
undergraduate work, Mr. Susan moved to Gainesville, Florida where he enrolled in the
Vertebrate Zoology Program of the College of Liberal Arts and Sciences at the
University of Florida and worked full-time as a laboratory technician in the Departments
of Zoology, Botany, and Biological Sciences, providing support for research and
teaching. During this work, he prepared technical illustrations published in laboratory
manuals for the University of Florida.
In 1988, Peter Susan then successfully completed a Masters Fellowship for
Teachers in the Department of Instruction and Curriculum of the College of Education at
the University of Florida, earning a master of education degree in science instruction and
curriculum. For several years, he served as a public school science teacher in Manatee
County, Florida.
By spring of 1992, Mr. Susan pursued and acquired a research assistantship in
the Department of Anatomy and Cell Biology of the College of Medicine at the
University of Florida. In 1993, he began studying protein turnover in the laboratory of
Dr. William A. Dunn, Jr. This dissertation resulted from much of that work.
176

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.
%
William A. Dunn, Jr., Chair
Associate Professor of Anatomy and
Cell 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 quality, as a dissertation for
the degree of Doctor of Philosophy.
Brian D. Cain
Associate Professor of Biochemistry
and Molecular 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 quality, as a dissertation for
the degree of Doctor of Philosophy.
K
'Mohan K. Raizada
Professor of Physiology
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.
Carl M. Feldherr
Professor of Anatomy and Cell
Biology
This dissertation was submitted to the Graduate Faculty of the College of Medicine and to
the Graduate School and was accepted as partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
August 1998
Dean, Graduate School

UNIVERSITY OF FLORIDA
lililll
3 1262 08555 3039



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INGEST IEID EHCPXES9J_CMMTKZ INGEST_TIME 2014-06-23T22:09:50Z PACKAGE AA00022295_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
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