Research
Interaction of Hsp70 chaperone and its co-chaperones in S. cerevisiae
The objective of our research is to elucidate the functional role of Hsp70 molecular chaperone. It is generally believed that Hsp70 (and its cognate Hsc70) participates in a number of cellular processes, including folding of protein, assembly protein complexes, and protein transport across membranes. Now, it is also clear that Hsc70 must work with other cellular proteins to exert the chaperoning functions. In the past, we carried out biochemical characterization of Hsc70, and have identified a subset of tetratricopeptide repeat (TPR) - containing proteins as Hsc70 - interacting proteins. Further analysis shows that the TPR domains in these proteins interact with the C-terminal PTIEEVD sequence of Hsc70. However, the C-terminal tail in Hsc70 is dispensable for its in vitro refolding activity. It therefore raises the possibility that Hsc70 might have the capacity to regulate the function of these TPR - proteins. If this were the case, besides chaperoning, Hsc70 should have some other as yet ill-defined functions.
Because of the limit of in vitro biochemical studies, we decided to use Saccharomyces cerevisiae as the experimental system to investigate the function in vivo of Hsp70 chaperone as well as its interacting partners. Specifically, we wish to address the following questions: (1) if one can correlate the reduction of Hsp70¡¦s refolding activity with the phenotypes of existing hsp70 mutants, (2) if disruption of the interaction between Hsp70 with the TPR - proteins may have some phenotypes, (3) whether chaperones are involved in as yet unknown cellular processes; and if so, what the chaperone¡¦s role is.
In S. cerevisiae, the four SSA genes (SSA1 - SSA4) are the equivalent of Hsp70/Hsc70 in vertebrates. We have generated SSA1 mutants with other SSA genes deleted in yeast with S288C background. Unfortunately, these mutants had the tendency to lose mitochondrial DNA. It makes the analysis difficult. Therefore, we are generating these mutants in yeast with a different genetic background (W303). We then shall carry out the phenotype analysis.
While there is a large number of TPR - containing proteins in yeast, only a small fraction of them (including Sgt2) possess the conserved residues at specific locations in the TPR motifs that mediate the interaction with Hsc70. Sgt2 is thought to be the homologue of vertebrate SGT. Since we previous performed biochemical analysis on SGT, we chose Sgt2 for further characterization. It appears that Sgt2 interacts with Ssa1/Ssa2 (presumably through its TPR domain). The N-terminal domain of Sgt2 interacts in vitro with Mdy2. MDY2 shows a synthetic interaction with YDJ1. (Ydj1 is another chaperone known to work together with Ssa1/Ssa2.) In addition, purified Mdy2 has the capacity to precipitate Ydj1, although it is not ruled out that this interaction is mediated by other proteins. Therefore, it is likely that the protein complexes composed of Sgt2 and Mdy2 (perhaps some other proteins) may bring different chaperones together to exert their chaperoning function.
Examination of the yeast data base reveals that chaperones interact genetically with some DNA synthesis and repair enzymes. We recently discovered that deletion of YDJ1 resulted in meiosis defect. However, through the analysis of YDJ1/SPO11 double deletion mutants, it appears that the defect might not be originated from DNA replication or recombination events. Thus, it remains to be determined what role of Ydj1 chaperone in meiosis is.
