Purification and biochemical properties of Saccharomyces cerevisiae's Mge1p, the mitochondrial cochaperone of Ssc1p.
Previous biochemical and genetic studies have demonstrated the universal conservation of the DnaK (Hsp70) chaperone machine. Its three members, DnaK, DnaJ, and GrpE, in Escherichia coli work synergistically to promote protein protection, disaggregation, and import into the various organelles. In the mitochondria of Saccharomyces cerevisiae the three corresponding members are designated as Ssc1p, Mdj1p, and Mge1p, respectively. The MGE1 gene was previously cloned by us and others, and its product has been shown to be absolutely essential for protein transport into mitochondria and hence cell viability. To better understand its biological role, we have proceeded to overexpress and purify the mature Mge1p in E. coli through the construction of the appropriate vector clone. Mge1p has been shown to functionally substitute for its E. coli GrpE counterpart in a variety of its biological functions, including suppression of the bacterial temperature-sensitive phenotype of the grpE280 mutation, formation of a stable complex with DnaK, stimulation of DnaK's ATPase activity, and the refolding of denatured luciferase by the DnaK/DnaJ chaperone proteins. Thus, the function of the GrpE homologues appears to be highly conserved across the biological kingdoms.
Deloche
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Département de Biochimie Médicale, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Geneva 4, Switzerland.
Georgopoulos
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000502:physiology; D001692:Biological Transport; D000252:Calcium-Transporting ATPases; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005800:Genes, Fungal; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
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; C086039:MGE1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D000252:Calcium-Transporting ATPases