Maintenance of protein homeostasis in eukaryotes under normal growth and stress conditions requires the functions of Hsp70 chaperones and associated co-chaperones. Here, we investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phosphomimetic substitutions at this site (S151D) dramatically downregulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phosphomimetic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 cochaperones Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phosphomimetic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [PSI+] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.
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