The long term objective is to gain insight into the role of the cytoplasmically located stress proteins in cellular processes and to better understand the regulation of expression of the genes that encode these proteins especially during exponential growth. More specifically we propose to study, using a combination of genetic and biochemical techniques, the function of the essential SSA subfamily of genes of Saccharomyces cerevisiae which is composed of 4 genes, SSAI, SSA2, SSA3 and SSA4, each encoding an hsp7O protein. The functional relationship between members of this structurally and functionally related family of genes will be determined. Whether they are functionally identical or distinct will be studied, by analyzing strains containing mutations in different SSA genes and expressing differing amounts of particular members of the subfamily. Point mutations in SSAL will be isolated and the phenotypes of strains carrying these mutations, including protein translocation in the endoplasmic reticulum (ER) and mitochondria, proteolysis, endocytosis and regulation of the heat shock response will be analyzed to gain a better understanding of the roles of SSA proteins in these processes. Second-site revertants of SSA mutations will be isolated to gain information about interacting proteins and processes in which the SSA genes are involved. We will clone and analyze the genes which are capable of suppressing the effects of mutations in SSA genes. The function of SSAL will be studied, using as a model system the translocation of precursor proteins into isolated mitochondria and microsomes. In these studies the SSAI mutants that have been isolated as well as the second-site suppressor mutations will be used to gain a better understanding of the role played by SSAl in protein translocation. The expression of SSA1, particularly during normal exponential growth will be studied to better understand the mechanism of regulation of basal as well as stressed-induced expression. We have identified an upstream repressing sequence (URS) that represses expression of SSAL. Mutants that are defective in URS-based repression will be isolated and analyzed in hopes of isolating mutants containing mutations in the gene that encodes the URS-binding protein. The mechanism of action of the URS-based repression will be analyzed using in vitro methods and the interaction of the heat shock transcription factor with a natural promoter both before and after a heat shock will be studied.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031107-13
Application #
2176019
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-07-01
Project End
1995-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
13
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Sahi, Chandan; Kominek, Jacek; Ziegelhoffer, Thomas et al. (2013) Sequential duplications of an ancient member of the DnaJ-family expanded the functional chaperone network in the eukaryotic cytosol. Mol Biol Evol 30:985-98
Kominek, Jacek; Marszalek, Jaroslaw; Neuvéglise, Cécile et al. (2013) The complex evolutionary dynamics of Hsp70s: a genomic and functional perspective. Genome Biol Evol 5:2460-77
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