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The Ydj1 molecular chaperone facilitates formation of active p60v-src in yeast
B Dey1, A J Caplan, F Boschelli
1Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Abstract:
Molecular chaperones have been implicated in the formation of active p60v-src tyrosine kinase. In Saccharomyces cerevisiae, expression of p60v-src causes cell death, a phenomenon that requires functional Hsp90. We show here that mutations in a member of a second class of chaperones, the yeast dnaJ homologue YDJ1, suppress the lethality caused by p60v-src. One p60v-src-resistant ydj1 mutant, ydj1-39, which has two point mutations in the highly conserved "J" domain, has reduced levels of v-src mRNA and protein. However, a ydj1 null mutant produces normal quantities of active p60v-src, indicating that Ydj1p facilitates, but is not essential for, the formation of active p60v-src. We also report p60v-src-resistance in a previously identified temperature-sensitive ydj1 mutant, ydj1-151. In this mutant, the level of p60v-src remains unaltered, but the protein is much less active in vivo. In addition, p60v-src immunoprecipitates from the ydj1-151 strain contained Hsp90 and Hsp70 in greater amounts than in wild-type strains. Ydj1 protein was also detected in p60v-src immunoprecipitates from both wild-type and ydj1-151 strains. These results indicate that Ydj1p participates in the formation of active p60v-src via molecular chaperone complexes.
Insights
Yeast dnaJ homologue YDJ1 (Ydj1p) assists in forming active p60v-src tyrosine kinase. While not essential, Ydj1p
Area of Science:
- Molecular biology
- Cellular biology
- Biochemistry
Background:
- Molecular chaperones, including Hsp90, are crucial for active p60v-src tyrosine kinase formation.
- Expression of p60v-src in Saccharomyces cerevisiae leads to cell death, dependent on Hsp90 function.
Purpose of the Study:
- To investigate the role of the yeast dnaJ homologue YDJ1 (Ydj1p) in the formation and activity of p60v-src.
- To determine if Ydj1p is essential or facilitative for p60v-src function and associated cellular effects.
Main Methods:
- Utilizing Saccharomyces cerevisiae strains with mutations in YDJ1 (ydj1-39, ydj1-151) and a ydj1 null mutant.
- Assessing p60v-src mRNA and protein levels, kinase activity in vivo, and co-immunoprecipitation with chaperones Hsp90 and Hsp70.
Main Results:
- Mutations in YDJ1 suppress p60v-src-induced lethality.
- A specific ydj1 mutant (ydj1-39) showed reduced p60v-src levels, while a null mutant had normal active p60v-src, indicating Ydj1p facilitates but is not essential.
- Another ydj1 mutant (ydj1-151) exhibited reduced p60v-src activity in vivo, with increased Hsp90 and Hsp70 association, and Ydj1p was detected in p60v-src complexes.
Conclusions:
- Ydj1p plays a facilitative role in the formation of active p60v-src tyrosine kinase.
- Ydj1p functions within molecular chaperone complexes, likely involving Hsp90 and Hsp70, to modulate p60v-src activity.