Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1.
Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.
Sakuragi
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S
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Liu
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Q
Craig
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E
D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D000252:Calcium-Transporting ATPases; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D008156:Luciferases / Q000378:metabolism; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; C086039:MGE1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D008156:Luciferases; D000252:Calcium-Transporting ATPases