The Function of the Yeast Molecular Chaperone Sse1 Is Mechanistically Distinct from the Closely Related Hsp70 Family

Lance Shaner, Amy Trott, Jennifer L. Goeckeler, Jeffrey L. Brodsky, Kevin A. Morano
2004 Journal of Biological Chemistry  
The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-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
more » ... in were assayed for their ability to complement sse1⌬ yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1⌬ and lethality of sse1⌬sse2⌬ cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1⌬ growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70. Cells respond to heat shock by induction of a specific set of genes that allow them to cope with and recover from stress. Many of these heat shock proteins (HSPs) 1 function as molecular chaperones, binding unfolded proteins and preventing aggregation or facilitating their refolding (1). One of the most well studied classes of chaperones is the Hsp70 family. Saccharomyces cerevisiae possesses 14 Hsp70 homologs, distributed in the cytosol, mitochondria, and endoplasmic reticulum (2). Hsp70s promote folding of nascent polypeptides, facilitate translocation of proteins across membranes, and protect the
doi:10.1074/jbc.m313739200 pmid:15028727 fatcat:ygto7sfhpva55pmampgnf54oze