Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli

Abstract

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°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.

The NH2-terminal Domain of the Chloroplast GrpE Homolog CGE1 Is Required for Dimerization and Cochaperone Function in Vivo

“…5B). OD212 carries both a deletion of the grpE gene and the compensatory dnaK332 allele and is incapable of growth at temperatures above 30°C (32). As judged from their ability to grow normally at 23°C, (over)expression of the six CGE1 derivatives appeared not to have negative effects on cell viability (Fig.…”

The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90C

“…As shown in Figure 6B, the bulk of the untreated recombinant proteins displayed the same migration pattern as the native proteins, i.e. HSP90C, HSP70B, and CDJ1 roughly comigrated with the 140-kD marker protein and CGE1 with the 66-kD Temperature-sensitive E. coli strain OD259 carrying a deletion of its dnaJ gene (Deloche et al, 1997) was transformed with a plasmid vector for the expression of mature, hexa-His-tagged CDJ1 (pCDJ1, pMS458) or the empty expression vector (pCDF). Dilutions of transformant cultures were spotted onto Luria-Bertani plates and incubated overnight at 23°C, 37°C, or 40°C.…”

“…To test whether also CDJ1 was able to functionally replace E. coli DnaJ, we introduced the pCDF expression vector alone, or the same vector encoding CDJ1 with an N-terminal hexa-His tag into E. coli strain OD259 carrying a deletion of the dnaJ gene (Deloche et al, 1997). As shown in Figure 4, OD259 containing the empty vector grew well at 23°C, but growth was impaired at 37°C and abolished at 40°C.…”

“…It was reported previously that the yeast mitochondrial DnaJ homolog Mdj1p and the Arabidopsis mitochondrial DnaJ homolog AtJ1 (now termed atDjB1) could at least partially complement the temperaturesensitive phenotype of an E. coli DdnaJ mutant strain (Kroczynska et al, 1996;Deloche et al, 1997). To test whether also CDJ1 was able to functionally replace E. coli DnaJ, we introduced the pCDF expression vector alone, or the same vector encoding CDJ1 with an N-terminal hexa-His tag into E. coli strain OD259 carrying a deletion of the dnaJ gene (Deloche et al, 1997).…”

“…pMS458 and the empty pCDF vector were transformed into temperaturesensitive E. coli strain OD259 containing a deletion of its dnaJ gene (Deloche et al, 1997). For gene expression from the T7 promoter of pCDF, an additional vector encoding T7 polymerase (Gentaur) was cotransformed.…”