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Lance Shaner

Alumnus

Earned Juris Doctor, Law at The University of Houston Law School after receiving PhD
Now Owner of Omega Yeast Labs L.L.C., Chicago

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Mutant Sse1 proteins with a truncation of up to 44 carboxyl-terminal residues not included in the PBD were active. Remarkably, the two domains of Sse1 when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication.

Relatively little is known regarding the interactions and cellular functions of Sse1. Through co-immunoprecipitation analysis, we found that Sse1 forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Sse1 and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Sse1 stimulated Ssa1 ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. This enzymatic activity is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals.

Cells lacking SSE1 were found to accumulate prepro-alpha-factor, but not the co-translationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Sse1 and Ssa is functionally significant in vivo. In addition, sse1Δ cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase Slt2. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.

Search pubmed for papers by L. Shaner and K. Morano

Research Info

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1