The ubiquitin (Ub)-proteasome system is responsible for the majority of intracellular protein degradation in eukaryotes. Both specific regulatory proteins and a broad range of misfolded or otherwise abnormal proteins must be recognized. Many diseases, including neurodegenerative disorders, diabetes, cystic fibrosis, and many types of cancer, are associated with abnormalities in the Ub system. Therefore, an understanding of how proteolytic substrates are recognized and covalently modified by Ub is expected to have a substantial impact on our ability to diagnose and treat many serious human medical conditions. In this renewal, a series of basic studies on Ub-dependent proteolysis is proposed. The focus is on the prototype natural substrate Mata2, a transcriptional regulator in the yeast S. cerevisiae. Mata2 is ubiquitinated in vivo by two distinct mechanisms. One depends on the Ubc6 and Ubc7 Ub-conjugating enzymes (E2s) and the Doa10 Ub ligase (E3), and the other on the Ubc4 E2. An Ubc6-Ubc7-Doa10 ubiquitination complex is believed to assemble on the endoplasmic reticulum (ER) or nuclear envelope (NE) and to recognize Mata2 through a Mata2 domain called the Deg1 degron. Neither the degron nor the E3 for the Ubc4 pathway is known; experiments are proposed in this application to identify both. In the remainder of the application, emphasis is placed on the transmembrane Doa10 Ub ligase, which targets both the soluble, nuclear Mata2 protein and ER membrane proteins. The overall goal is to define the key biochemical and cell biological determinants of specificity for this conserved ER/NE membrane-embedded Ub-ligation system. Evidence for a major role for Doa10 and its orthologs in ER physiology is beginning to emerge, so these experiments are expected to shed light on ER-associated and nuclear protein degradation in organisms ranging from yeast to human.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-PBC (02))
Program Officer
Ikeda, Richard A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Yale University
Schools of Medicine
New Haven
United States
Zip Code
Hickey, Christopher M; Xie, Yang; Hochstrasser, Mark (2018) DNA binding by the MAT?2 transcription factor controls its access to alternative ubiquitin-modification pathways. Mol Biol Cell 29:542-556
Budenholzer, Lauren; Cheng, Chin Leng; Li, Yanjie et al. (2017) Proteasome Structure and Assembly. J Mol Biol 429:3500-3524
Huber, Eva M; Heinemeyer, Wolfgang; Li, Xia et al. (2016) A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat Commun 7:10900
Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark (2016) Substrate specificity of the ubiquitin and Ubl proteases. Cell Res 26:441-56
Zattas, Dimitrios; Berk, Jason M; Kreft, Stefan G et al. (2016) A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation. J Biol Chem 291:12105-18
Li, Xia; Li, Yanjie; Arendt, Cassandra S et al. (2016) Distinct Elements in the Proteasomal ?5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly. J Biol Chem 291:1991-2003
Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark (2016) Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells. Cell Rep 14:2962-74
Zattas, Dimitrios; Hochstrasser, Mark (2015) Ubiquitin-dependent protein degradation at the yeast endoplasmic reticulum and nuclear envelope. Crit Rev Biochem Mol Biol 50:1-17
Hickey, Christopher M; Hochstrasser, Mark (2015) STUbL-mediated degradation of the transcription factor MAT?2 requires degradation elements that coincide with corepressor binding sites. Mol Biol Cell 26:3401-12
Kunjappu, Mary J; Hochstrasser, Mark (2014) Assembly of the 20S proteasome. Biochim Biophys Acta 1843:2-12

Showing the most recent 10 out of 54 publications