The Etp1 Ubiquitin Ligase Regulates
Ubiquitin Homeostasis in Yeast
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
Background and Introduction
The Ethanol Tolerance Protein 1 Ubiquitin Ligase
Regulates Ubiquitin Homeostasis in Yeast
Yeast-Two-Hybrid Analysis of BRAP2 Binding
Conclusions and Future Directions
List of tables and figures
The ubiquitination pathway.
The ubiquitin conjugation cascade.
Different E2s may be required for substrate ubiquitination.
HECT and RING E3 ubiquitin ligases.
The RING finger domain.
RING E3s may use different mechanisms to catalyze
polyubiquitination of substrate.
Ubiquitin chain topology.
Potential sites for drug development in the ubiquitin-
proteasomal system (UPS).
Domain structure of BRAP2.
ZnF UBP domain.
Orthologues of BRAP2.
Plasmids used in this study.
Oligonucleotides used in this study.
S. cerevisiae strains used in this study.
Figure 1. Etp1 is a short-lived homologue of BRAP2.
Etp1 is an E3 ubiquitin ligase.
Etp1 binds ubiquitin.
Levels of free ubiquitin chains are lowered in etp1Δ strain.
Phenotypes of etp1Δ. 93
ETP1 and DOA4 act upon the same pathway.
A model of regulation of Ub homeostasis by Etp1 and
The two-hybrid principle.
Northern hybridization of BRAP2.
Alignment of CHCHD3 hits. 128
Classification of Y2H hits for BRAP2.
Reduction in abundance of highly expressed gene
transcripts following cDNA normalization.
Homology model of ETP1 and USP5 ZnF UBP domains.
Compete list of YH2 hits for screen 2.
A model of regulation of Ub homeostasis by
deubiquitinating enzymes and ubiquitin ligases.
Preliminary Genetic interactions of ETP1.
About this Dissertation
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|The Etp1 Ubiquitin Ligase Regulates Ubiquitin Homeostasis in Yeast ()||2018-08-28||