The objectives of the proposed research are to identify structural elements in short-lived proteins that cause them to be turned over rapidly, and to identify and characterize the enzymatic machinery involved in this process. The focus of the application is the transcriptional repressor protein MAT(2, which is turned over with a half-life of about 4 minutes in a ubiquitin-dependent manner.
Five specific aims are described. The first is to further define the molecular determinants that render the alpha2 repressor short-lived. Mutations in the N-terminal region of alpha2 (Deg1 signal) have been identified that inhibit turnover. These mutations will be used in a genetic selection/screen to identify trans-acting factors that enhance alpha2 degradation (edd genes). Experiments will also be done to identify the alpha2 sites of ubiquitination.
Aim #2 is to determine the mechanistic basis of cell-type specific regulation of alpha2 turnover. Alpha2 is more stable in diploid cells, where it functions as an a1/alpha2 complex, than in alpha cells. Experiments are proposed to test the hypothesis that alpha2 is stabilized through its interaction with a1. Reciprocal experiments are also proposed to determine whether stability of the a1 protein might be affected by interaction with alpha2.
Aim #3 is to further characterize doa genes, which were identified in a screen for mutants defective in alpha2 turnover. Previous genetic analysis defined 10 different doa genes. Four of these genes (DOA2 - DOA5) encode components of the ubiquitin-proteosome degradation pathway. In this aim experiment are proposed to further characterize the DOA1 and DOA2 (UBC6) genes and to clone and characterize the DOA6 - DOA10 genes.
The fourth aim i s to investigate the pathway of proteosome assembly. This work will focus on the DOA3 and PRE3 proteosomal subunits, which are synthesized as N-terminal precursor proteins. Pulse-chase experiments will be done to monitor proteasome biogenesis and the structural requirements for functional DOA3 and PRE3 will be determined using a plasmid-shuffle assay.
The final aim i s to investigate the structural basis of the multiple catalytic sites in the yeast proteasome and to determine the relative positions of proteasome subunits. These experiments will focus on DOA3 and PRE1, since alleles of either cause the proteasome to be defective in chymotrypsin-like activity. The experimental approach will focus on extragenic suppressors of defined subunit alleles and will be supplemented with chemical cross-linking studies.
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