PURIFICATION AND BIOCHEMICAL-PROPERTIES OF SACCHAROMYCES CEREVISIAES MGE1P, THE MITOCHONDRIAL COCHAPERONE OF SSC1P

Previous biochemical and genetic studies have demonstrated the univers al conservation of the DnaK (Hsp70) chaperone machine, Its three membe rs, DnaK, DnaJ, and GrpE, in Escherichia coli work synergistically to promote protein protection, disaggregation, and import into the variou s organelles, In the mitochondria of Saccharomyces cerevisiae the thre e corresponding members are designated as Ssc1p, Mdj1p, and Mge1p, res pectively, The MGE1 gene was previously cloned by us and others, and i ts product has been shown to be absolutely essential for protein trans port into mitochondria and hence cell viability, To better understand its biological role, we have proceeded to overexpress and purify the m ature Mge1p in E. coli through the construction of the appropriate vec tor clone. Mge1p has been shown to functionally substitute for its E. coli GrpE counterpart in a variety of its biological functions, includ ing suppression of the bacterial temperature-sensitive phenotype of th e grpE280 nutation, formation of a stable complex with DnaK, stimulati on of DnaK's ATPase activity, and the refolding of denatured luciferas e by the DnaK/DnaJ chaperone proteins. Thus, the function of the GrpE homologues appears to be highly conserved across the biological kingdo ms.