Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.
The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.
Deloche
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Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Kelley
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Georgopoulos
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D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D000252:Calcium-Transporting ATPases; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D005798:Genes, Bacterial; D005800:Genes, Fungal; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002352:Carrier Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C087041:MDJ1 protein, S cerevisiae; C086039:MGE1 protein, S cerevisiae; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C063842:dnaK protein, E coli; D000252:Calcium-Transporting ATPases