Areas of Interest
Our research focuses on the role of the ubiquitin system in protein degradation and sorting. Conjugation to ubiquitin is an obligatory step for protein degradation in eukaryotes.
Precise control of protein breakdown by the ubiquitin system is crucial for numerous cellular processes including cell cycle progression, cell growth and proliferation, signal
transduction, and transcription. This system has been implicated in several forms of malignancy, in the pathogenesis of several genetic diseases, in immune surveillance/viral
pathogenesis, and in chronic degenerative disorders of the nervous system.
Ubiquitination is a reversible process. Ubiquitin is a stable protein and, therefore, it has to be recycled by the action of DUBs prior to the degradation of ubiquitin-protein
conjugates. Many DUBs have been recently identified in eukaryotic organisms. However, relatively little is known about their physiological functions or natural substrates. Potentially,
DUBs may act as negative and positive regulators of the ubiquitin system. In addition to ubiquitin recylcing, they are involved in processing of ubiquitin precursors, in proofreading of
protein ubiquitination and in disassembly of inhibitory ubiquitin chains.
We use a simple eukaryote Saccharomyces cerevisiae as a model system because it provides the possibility, at a level not yet available in any other eukaryotic organism, of both
genetic manipulation and detailed biochemical analysis. Given the high degree of conservation of fundamental metabolic systems between yeast and higher eukaryotes in general, the results
of our research will provide important insights into selective protein degradation in other eukaryotes as well. Our long-term goal is to identify and to characterize particular members
of the yeast DUB family that are responsible for each step of ubiquitin homeostasis in the eukaryotic cell.
Lab Rotation Projects
Analysis of the yeast deubiquitinating enzymes*
#1 - Affinity purification of the yeast protein complexes containing deubiquitinating enzyme Doa4.
Doa4 is a critical player in ubiquitin homeostasis in the yeast Saccharomyces cerevisiae. We have found that Doa4 is involved in ubiquitin recycling at the 26S proteasome and at the
cytoplasmic face of the late endosome. We believe that the enzymatic activity of Doa4 must be thoroughly regulated. Otherwise, uncontrolled deubiquitination may have critical
consequences for eukaryotic cell. Conceivably, regulation of Doa4 occurs at the levels of interaction with protein cofactors. The major goal of this project is to identify Doa4 partners
in yeast. The results will be important for understanding the mechanistic details of how Doa4 functions in vivo.
#2 – Analysis of the molecular basis for genetic interaction between mutations in deubiquitinating enzyme Doa4and class E vacuolar protein sorting factors.
Inactivation of the class E vacuolar protein factors leads to efficient suppression of doa4 mutant phenotypes. To determine the mechanisms of this suppression, we plan to conduct a
mutant screen based on the mini-Tn3 transposon-mutagenized library. We expect that this approach will identify deubiquitinating enzymes that are involved, along with Doa4, in membrane
protein trafficking in yeast.
*Deubiquitinating enzymes are highly specific proteases that remove ubiquitin (a small protein that modifies substrate proteins) from ubiquitin-protein conjugates and are responsible
for processing of ubiquitin precursors.
Selected Publications
Amerik, AY, Hochstrasser, M. Mechanism and function of deubiquitinating enzymes. Biochem. et Biophys. Atca., (in press).
Amerik, AY, Akhtar, N., Hochstrasser, M. Two conserved motifs are essential for interaction of the Doa4 deubiquitinating enzyme with the late endosome. (manuscript in preparation).
Amerik, AY, Hochstrasser, M. 2004. The Saccharomyces cerevisiae deubiquitinating enzyme Doa4. In handbook of proteolytic enzymes, 2nd edition (Barrett, AJ, Rawlings, ND, Woessner, JF
eds) London: Academic Press.
Amerik, AY, Li, SJ, Hochstrasser, M. 2000. Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae. Biol Chem. Sep-Oct;381(9-10):981-92.
Amerik AY, Nowak J, Swaminathan S, Hochstrasser M. 2000. The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol Biol Cell.
Oct;11(10):3365-80. |
