Abstract
A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.
Highlights
Saccharomyces cerevisiae' has been used to comple- cancomplement a defined S. pombe mutant lacking this ment a mutant of Schizosaccharomyces pombelacking subunit.The expression of a functionalsubunitas docus. the subunit (Boutry, M., and Goffeau, A. (1982)Eur
Theco-transformation for LEU+and growth on glycerol transformed both S. cereuisiae and S. pombe mutants lacking medium was dependent on the presenceof the plasmid since afunctionalF,ATPase @ subunit
This Coomassie blue-st,ainedprotein of mitochondriaprepared indicates that the S. cereuisiae subunit is assembled into the
Summary
Andtheplasmid pJ14' which is recoveredfrom thetransformant grown oneither YG medium or SD-Leu.The arrows aroundthe. Theco-transformation for LEU+and growth on glycerol transformed both S. cereuisiae and S. pombe mutants lacking medium was dependent on the presenceof the plasmid since afunctionalF,-ATPase @ subunit. The cereuisiae genomic insert (Fig. 1).The 3.3-kb EcoRI fragment yield of cells in late stationary phase was 69% (cell number) designated E3 of pJ14-1 has been shown to contain approxi- and 78% (wet weight) as compared to the parenttaylpe These mately 1.5 kb of ATP2 coding DNA in addition to the5' and results suggest thattransformation of mutant B59-1with. Gof- which is expressed asa lower :'"P,-ATP exchange rate is feau, manuscript submitted
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