In this proposed study, we will test the hypothesis that the degradation of proteins by the ubiquitin-dependent pathway requires the intermediate formation of a unique multiubiquitin chain on a substrate protein. In this multiubiquitin chain, the C-terminal carboxyl group of one ubiquitin molecule is linked via an isopeptide bond to the e-amino group of lysine 48 of an adjoining ubiquitin molecule. Whether the degradation of a specific protein in the in vitro reticulocyte lysate system requires such a multiubiquitin chain intermediate can be tested initially by replacing wild-type ubiquitin in the degradative assay with a ubiquitin mutant, Ub-R48, in which lysine 48 was replaced by an arginine. Further structural studies can then be made to confirm the existence of this unique multiubiquitin chain in the degradative intermediates. The expression of Ub-R48 in the yeast Saccharomyces cerevisiae will also permit the testing of this hypothesis in vivo. In order to test the function of this multiubiquitin chain, we have also proposed to synthesize chemically a sufficient amount of an analog so that we may study its interaction with a purified protease which has been shown to act preferentially on ubiquitinated proteins. Positive results from these proposed studies should elucidate the mechanism by which proteolytic targeting is achieved by the posttranslational addition of ubiquitin to proteins. We also propose to characterize systematically the posttranslational addition of ubiquitin to proteins in the yeast nucleus. These characterizations will include the identification of nuclear ubiquitin conjugation enzymes and their substrates. Long term objectives in this research will include the cloning of the substrate proteins and studies of their functions by gene deletion and mutations. As part of our continuing effort to understand our finding of ubiquitin in the neurofibrillary tangles of Alzheimer's brain, we wish to determine how an insufficient level of cellular ubiquitin may cause cell death. Because the ubiquitin system is highly conserved among eukaryotes, relevant biochemical changes caused by an insufficient level of ubiquitin can be determined using the yeast Saccharomyces cerevisiae. Studies are proposed here to characterize the biochemical changes in a yeast mutant in which the polyubiquitin gene had been deleted.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035803-06
Application #
3289054
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Wayne State University
Department
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Banerjee, A; Gregori, L; Xu, Y et al. (1993) The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein. J Biol Chem 268:5668-75
Cook, W J; Jeffrey, L C; Xu, Y et al. (1993) Tertiary structures of class I ubiquitin-conjugating enzymes are highly conserved: crystal structure of yeast Ubc4. Biochemistry 32:13809-17
Haas, A L; Reback, P B; Chau, V (1991) Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. Comparison to their putative rabbit homologs, E2(20K) AND E2(32K). J Biol Chem 266:5104-12
Dunten, R L; Cohen, R E; Gregori, L et al. (1991) Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme. J Biol Chem 266:3260-7
Gregori, L; Poosch, M S; Cousins, G et al. (1990) A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis. J Biol Chem 265:8354-7
Chau, V; Tobias, J W; Bachmair, A et al. (1989) A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243:1576-83
Shaw, G; Chau, V (1988) Ubiquitin and microtubule-associated protein tau immunoreactivity each define distinct structures with differing distributions and solubility properties in Alzheimer brain. Proc Natl Acad Sci U S A 85:2854-8
Perry, G; Friedman, R; Shaw, G et al. (1987) Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. Proc Natl Acad Sci U S A 84:3033-6
Meyer, E M; West, C M; Chau, V (1986) Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes. J Biol Chem 261:14365-8