Silberg, Jonathan J.2011-07-252011-07-252010Zhai, Peng. "Escort protein regulation of the human mitochondrial molecular chaperone mtHsp70." (2010) Diss., Rice University. <a href="https://hdl.handle.net/1911/61973">https://hdl.handle.net/1911/61973</a>.https://hdl.handle.net/1911/61973Mitochondrial Hsp70 molecular chaperones (mtHsp70) are nuclear-encoded proteins that are needed for the import of proteins into the mitochondrial matrix, biogenesis of Fe/S-clusters involved in energy metabolism, and stress response folding. Unlike the Hsp70 homologs found in other subcellular compartments, two of the mtHsp70 homologs in yeast require a specialized Hsp70 escort protein (Hep1) to maintain their solubility and function. To better understand the role that escort proteins play in regulating their cognate chaperones, I have characterized the regulation of human mtHsp70 by the human Hep, an ortholog of Hep1. In my thesis research, I show that human Hep is localized to the mitochondria of tissue culture cells, like mtHsp70. In addition, I demonstrate that human Hep enhances the solubility of mtHsp70 upon overexpression in Escherichia coli through an interaction with its N-terminal ATPase domain (70ATPase). Chromatography, fluorescence, and copurification analysis using recombinant proteins indicate that Hep binds most tightly to nucleotide-free mtHsp70 and 70ATPase. While this complex is destabilized by the presence of ADP and ATP, ATPase measurements indicate that Hep binds to ATP-bound mtHsp70 (and 70ATPase) and stimulates the steady-state rate of ATP hydrolysis, implicating a role for Hep in directly regulating the mtHsp70 chaperone reaction cycle. To identify Hep residues that are critical for chaperone regulation, I also carried out an alanine mutagenesis scan of charged residues in a tryptophan-free mutant (W1151) of human Hep and assessed the effect of each mutation on Hep and mtHsp70 interactions. In vitro binding studies identified three mutations (R81A, H107A, and D111A) with decreased affinity to nucleotide-free chaperone, and ATPase measurements revealed that one of these mutants (H107A) fails to elicit an increase in the steady-state as activity of 70ATPase. Yeast complementation studies further revealed that Hep supports the growth of Deltahep1 Saccharomyces cerevisiae like yeast Hep1, whereas a Hep-H107A cannot complement Deltahep1 yeast. These findings demonstrate that human Hep is a functional ortholog of yeast Hep1, and provide the first evidence that escort proteins directly regulate the catalytic activity of nucleotide-bound chaperones. Furthermore, they identify a histidine conserved in all mitochondrial and plastid escort proteins as critical for the regulation of human mtHsp70.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.Molecular biologyChemistryBiochemistryThesisTHESIS BIOCHEM. 2010 ZHAI