The Saccharomyces cerecisiae hsp70 escort protein Hep1 exhibits cross-reactivity with a non-cognate chaperone

Abstract

Genetic studies have provided evidence that the hsp70 escort protein Hep1 is essential for maintaining the solubility of the mitochondrial chaperones Ssc1 and Ssq1 in Saccharomyces cerevisiae. However, little is known about its metal binding thermostability, and substrate specificity. To characterize these properties, I have demonstrated that the four conserved cysteines in the zinc-finger motif of Hep1 are required for solubility and zinc binding. Mutation of any one of these cysteines to serine abolished soluble expression in Escherichia coli. In addition, elemental analysis of purified Hep1 revealed near stoichiometric levels of zinc in purified Hep1. This zinc could be displaced by the sulfhydryl-reactive agent methyl methanethiosulfonate (MMTS) implicating the cysteines in the zinc motif as ligands for zinc. A possible interaction between yeast Hep1 and human mitochondrial hsp70 (mthsp70) was also examined by measuring the influence of Hep1 on the solubility of mthsp70 overexpressed in E. coli. Human mthsp70 was found predominantly in the soluble faction upon coexpression with yeast Hep1, whereas mthsp70 was completely insoluble in the absence of Hep1. This interaction is thought to be mediated by the mthsp70 ATPase domain since a mthsp70 truncation mutant having only its N-terminal ATPase domain was insoluble when overexpressed in E. coli. To examine whether Hep1 influences mthsp70 thermostability, the effects of temperature on the CD spectra of purified mthsp70 and Hep1 were investigated. Hep1 exhibited a high melting temperature (Tm > 70°C), whereas mthsp70 displayed two transitions (Tm 1 48°C; Tm2 78°C), which were not dramatically altered in the presence of Hep1. Additional studies characterized the solubility and metal content of the small J-type co-chaperone, HscB, which is thought to regulate the ATPase activity of mthsp70. Unlike yeast HscB orthologs, human HscB contains a domain with a tetracysteine motif. Human HscB was shown to be localized to the mitochondria and was found to be soluble when overexpressed in E. coli. Magnetospirillum magneticum HscB, which contains an N-terminal Cys-rich domain similar to that of human HscB, was shown to coordinate an iron cofactor in an oxygen-sensitive manner. These findings provide evidence that Hep1 requires bound zinc to maintain its solubility, that the hsp70 ATPase domain is responsible for the low solubility of mthsp70, and implicate a role for escort proteins in regulating the functions of metazoan chaperones. Additionally, they implicate a role for the Cys-rich domain of HscB in metal coordination.