Abstract
In yeast, Hsp70 escort protein Hep1 regulates the solubility and functions of mitochondrial molecular chaperones, which are essential for protein folding, protein translocation, and Fe/S‐cluster biogenesis. Little is known about why escort proteins have evolved to regulate chaperone functions and whether human mitochondrial Hsp70 (mtHsp70) also requires escort proteins. We have examined the metal content, stability, and substrate specificity of Saccharomyces cerevisiae Hep1 (ScHep1). Elemental analysis revealed that Hep1 contains stoichiometric levels of zinc, and mutation of any one of its four conserved cysteines to serine abolished Hep1 solubility upon expression in Escherichia coli. In vitro analysis of Hep1 solubility further showed that removal of zinc by the strong oxidant methanethiolsulphonate led to aggregation, whereas zinc‐bound Hep1 remained soluble when heated from 20 to 70°C and exhibited little change in its circular dichroism spectrum. A possible interaction between yeast Hep1 and human mtHsp70 was also examined by measuring the influence of Hep1 on the solubility of mtHsp70. When expressed alone in Escherichia coli, mtHsp70 was insoluble. However, coexpression with Hep1 enhanced the level of soluble mtHsp70 produced. Furthermore, gel filtration analysis revealed that mtHsp70 self‐associates in the absence of nucleotides, and dissociates into monomers when incubated with Hep1 or ATP. Ongoing work is investigating whether human Hep has similar in vivo and in vitro functions as yeast Hep. These findings provide evidence that Hep1 requires bound zinc to maintain its solubility, and they implicate a role for escort proteins in regulating the oligomerization of human chaperones. They also suggest that Hep homologs may adjust chaperone solubility and function in response to biological oxidants.
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