Ubiquitin-Like Small Archaeal Modifier Proteins in the Haloarchaeon Haloferax volcanii

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Material Information

Title:
Ubiquitin-Like Small Archaeal Modifier Proteins in the Haloarchaeon Haloferax volcanii
Physical Description:
1 online resource (200 p.)
Language:
english
Creator:
Miranda, Hugo Vinicio
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Microbiology and Cell Science
Committee Chair:
MAUPIN,JULIE A
Committee Co-Chair:
ROMEO,TONY
Committee Members:
SHANMUGAM,KEELNATHAM T
GONZALEZ,CLAUDIO F
CHEN,SIXUE

Subjects

Subjects / Keywords:
archaea -- proteasome -- ubiquitin
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre:
Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The genome of Haloferax volcanii encodes three ubiquitin-like small archaeal modifier proteins (SAMPs)that function as protein modifiers (SAMP1/2/3) or potentially as sulfur carriers (SAMP1/2). In this study, it was determined that SAMP1/2/3 function as protein modifiers. SAMPs were found to form protein conjugates that required their C-terminal diglycine motif for attachment. Additionally, the start codon for SAMP3 was determined. The E1-like protein UbaA was identified as being important for the conjugation of SAMP1/2/3 onto substrates. Deletion of ubaA prevented the attachment of SAMPs onto substrates and expression of ubaA in trans could restore SAMP conjugation. No other genes investigated in this study were required for the attachment of SAMPs onto their substrates. SAMP1 and SAMP2 conjugates were found to accumulate when Hfx. volcanii cells were treated with the proteasome inhibitor Velcade, suggesting that SAMP1 and SAMP2 act as proteolytic signals within the cell. SAMP3 conjugate levels,on the other hand, were not altered when cells were treated with Velcade, suggesting that SAMP3 has a non-proteolytic function in Hfx. volcanii. To definitively show that SAMPs form covalent bonds with protein targets, tandem mass spectrometry (MS/MS) analysis was used to map isopeptide bonds formed between the C-terminal glycine of SAMP2/3 and the epsilon-amino group of lysine residues on protein targets. In addition to their role as protein modifiers, SAMPs were studied to determine their roles in sulfur transfer pathways. In particular, work focused on determining if SAMP1 and SAMP2 are involved in molybdenum cofactor (MoCo) biosynthesis and tRNA thiolation, respectively. Data suggest that SAMP1, UbaA and MoaE may play a role in MoCo biosynthesis since a samp1, ubaA or moaE deletion mutant cannot grow anaerobically using DMSO as the terminal electron acceptor. SAMP2 and UbaA were found to be important for tRNA thiolation. The samp2 and ubaA deletion mutants have lower levels of thiolated tRNA compared to Hfx. volcanii wild type cells. In addition, samp2 and ubaA deletion strains display a high temperature sensitivity phenotype that is common among tRNA modification proteins. Overall, our data suggest that SAMPs are important for various cellular processes.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Hugo Vinicio Miranda.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: MAUPIN,JULIE A.
Local:
Co-adviser: ROMEO,TONY.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046287:00001