The Etp1 Ubiquitin Ligase Regulates Ubiquitin Homeostasis in Yeast Öffentlichkeit

Abstract

The Etp1 Ubiquitin Ligase Regulates
Ubiquitin Homeostasis in Yeast

By
Janetta A. Bryksin

Ubiquitination is a reversible post-translational modification of cellular proteins that regulates a wide array of cell processes and is implicated in many diseases. Although ubiquitin is a highly abundant protein, it is not produced in excess. Rather, there is a dynamic equilibrium between three forms of cellular ubiquitin: monomeric ubiquitin, a substrate-conjugated mono- and polyubiquitin, and unanchored ubiquitin chains. Maintenance of ubiquitin homeostasis is a tightly regulated process and involves several regulatory mechanisms.
Here, we characterize the role of Ethanol Tolerance Protein 1 or ETP1 in the regulation of ubiquitin homeostasis. S. cerevisiae Etp1 is a putative homologue of the human BRCA1 Associated Protein 2 or BRAP2. BRAP2 is a cytoplasmic E3 ubiquitin ligase that modulates the sensitivity of MAP kinase cascade. Also, BRAP2 binds to the NLS motif of various proteins and sequesters them in the cytoplasm. Our studies indicate that Etp1 has the same functional domains as BRAP2, which allows using a yeast model system to study the function of BRAP2.
ETP1 was previously described to play role in yeast adaptation to ethanol provided either as a sole carbon source or as a stressor. We hypothesize that, in addition to this function, ETP1 is implicated in the regulation of the ubiquitin homeostasis by mediating the formation of free ubiquitin chains. Upon loss of ETP1, yeast cells exhibit a decreased amount of polyubiquitin without changes in total ubiquitin levels. This function is dependent on the E3 ligase activity and the ubiquitin binding capability of Etp1. Loss of ETP1 leads to resistance of yeast to various stresses such as oxidative stress, translational inhibition, and an amino acid analog. Analysis of topology of ubiquitin chains catalyzed by Etp1 and its corresponding E2, Ubc4, has revealed the formation of K6, K11, K33, K48, and K63 ubiquitin chains. Doa4 is a deubiquitinating enzyme required for ubiquitin homeostasis. Deletion of ETP1 from doa4Δ yeast strain partially rescues the phenotypes of doa4Δ, indicating that ETP1 and DOA4 may act upon the same biological pathway. We therefore predict that Etp1 may be a ubiquitin sensor in a cell, controlling the pool of free ubiquitin chains.

Table of Contents

Table of contents

Page
CHAPTER I
Background and Introduction
1




CHAPTER II
The Ethanol Tolerance Protein 1 Ubiquitin Ligase
Regulates Ubiquitin Homeostasis in Yeast
57



CHAPTER III
Yeast-Two-Hybrid Analysis of BRAP2 Binding
Partners
114



CHAPTER IV
Conclusions and Future Directions
145

List of tables and figures

Chapter I

Page
Figure 1.
The ubiquitination pathway.
4
Figure 2.
The ubiquitin conjugation cascade.
5
Figure 3.
Different E2s may be required for substrate ubiquitination.
8
Figure 4.
HECT and RING E3 ubiquitin ligases.
11
Figure 5.
The RING finger domain.
13
Figure 6.
RING E3s may use different mechanisms to catalyze
polyubiquitination of substrate.
15
Figure 7.
Ubiquitin chain topology.
19
Figure 8.
Ubiquitin homeostasis.
24
Figure 9.
Potential sites for drug development in the ubiquitin-
proteasomal system (UPS).
26
Figure 10.
Domain structure of BRAP2.
31
Figure 11.
ZnF UBP domain.
33
Figure 12.
Orthologues of BRAP2.
35
Chapter II

Table 1.
Plasmids used in this study.
72
Table 2.
Oligonucleotides used in this study.
74
Table 3.
S. cerevisiae strains used in this study.
76
Figure 1.
Figure 1. Etp1 is a short-lived homologue of BRAP2.
79

Figure 2.

Etp1 is an E3 ubiquitin ligase.
83
Figure 3.
Etp1 binds ubiquitin.
87
Figure 4.
Levels of free ubiquitin chains are lowered in etp1Δ strain.
90
Figure 5.
Phenotypes of etp1Δ. 93
Figure 6.
ETP1 and DOA4 act upon the same pathway.
97
Figure 7.
A model of regulation of Ub homeostasis by Etp1 and
Doa4. 103
Chapter III

Figure 1.
The two-hybrid principle.
118
Figure 2.
Northern hybridization of BRAP2.
125
Figure 3.
Alignment of CHCHD3 hits. 128
Table 1.
Classification of Y2H hits for BRAP2.
130
Figure 4.
Reduction in abundance of highly expressed gene
transcripts following cDNA normalization.
132
Figure 5.
Homology model of ETP1 and USP5 ZnF UBP domains.
134
Appendix I.
Compete list of YH2 hits for screen 2.
139
Chapter IV

Figure 1.
A model of regulation of Ub homeostasis by
deubiquitinating enzymes and ubiquitin ligases.
158
Appendix I
Preliminary Genetic interactions of ETP1.
169

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular
Stichwort	Etp1, ubiquitin homeostasis, yeast
Committee Chair / Thesis Advisor	Wilkinson, Keith D, Emory University
Committee Members	Price, Stephen Russ, Emory University Vertino, Paula M, Emory University Lynn, David, Emory University Corbett, Anita, Emory University Peng, Junmin, Emory University

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