Previous studies from Dr. Finley's laboratory defined the 17 subunits of the yeast proteasome regulatory particle. These samples had been exposed to high salt during conventional purification. Recently an affinity-purification method for proteasomes was developed by Dr. Finley's laboratory. Proteins from the salt wash fraction of the purification were identified by mass spectrometry and subsequently shown to be bona fide proteasome-associated proteins (PAPs). These appear to be novel in vivo components of the proteasome; some appear to be approximately stoichiometric. The new components are a ubiquitin-protein ligase (Hul5), a deubiquitinating enzyme (Ubp6), a protein that stabilizes the association of the proteasome core particle and the regulatory particle (Ecru29), and a protein that has been implicated in DNA repair (Bkn3). Each of these four genes is co-regulated with genes for known proteasome subunits, and strongly induced by the alkylating agent MMS. In addition, the known PAP proteins are all evolutionarily conserved. Dr. Finley proposes to define the complete set of major PAPs, and to study the roles of these proteins in proteasome function in vivo and in vitro. Sequences responsible for proteasome binding will be mapped within each PAP, and the phenotypic consequences of the loss of proteasome association will be determined. The spectrum of proteasome substrates stabilized by loss of function mutations in PAP genes will be assessed globally using powerful new mass spectrometry methods. Loosely associated cofactors are critical for the functioning of many protein complexes, such as polymerases, ribosomes, microtubules, and nuclear pores. Therefore, systematic investigation of proteasome-associated proteins may significantly enhance our understanding of this important protein complex.
Specific Aims are as follows:
Aim 1. To define the major proteasome-associated proteins.
Aim 2. To search globally for degradation defects in PAP mutants by mass spectrometry.
Aim 3. To localize within the proteasome the binding sites for specific associated proteins.
Aim 4. To map sequences within proteasome-associated proteins that mediate their binding to proteasomes.
Aim 5. To study the functions of specific proteasome-associated proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry Study Section (BIO)
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Ikeda, Richard A
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Harvard University
Anatomy/Cell Biology
Schools of Medicine
United States
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Finley, Daniel; Chen, Xiang; Walters, Kylie J (2016) Gates, Channels, and Switches: Elements of the Proteasome Machine. Trends Biochem Sci 41:77-93
Schmidt, Marion; Finley, Daniel (2014) Regulation of proteasome activity in health and disease. Biochim Biophys Acta 1843:13-25
Tar, Krisztina; Dange, Thomas; Yang, Ciyu et al. (2014) Proteasomes associated with the Blm10 activator protein antagonize mitochondrial fission through degradation of the fission protein Dnm1. J Biol Chem 289:12145-56
Chen, Ping-Chung; Bhattacharyya, Bula J; Hanna, John et al. (2011) Ubiquitin homeostasis is critical for synaptic development and function. J Neurosci 31:17505-13
Lee, Min Jae; Lee, Byung-Hoon; Hanna, John et al. (2011) Trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes. Mol Cell Proteomics 10:R110.003871
Park, Soyeon; Tian, Geng; Roelofs, Jeroen et al. (2010) Assembly manual for the proteasome regulatory particle: the first draft. Biochem Soc Trans 38:6-13
Isasa, Marta; Katz, Elijah J; Kim, Woong et al. (2010) Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome. Mol Cell 38:733-45
Lee, Byung-Hoon; Lee, Min Jae; Park, Soyeon et al. (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467:179-84
Finley, Daniel (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477-513
Hanna, John; Meides, Alice; Zhang, Dan Phoebe et al. (2007) A ubiquitin stress response induces altered proteasome composition. Cell 129:747-59

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