ARCHIVES OFBIOCHEMISTRYANDBIOPHYSICS Vol. 203, No. 1, August, pp. 260-270, 1980 Biogenesis of Mitochondria: A Temperature Sensitivity Mutation Affecting tt Mitochondrially Synthesized varl Protein of Saccharomyces cerevisiael MARK MURPHY, KONG-BUNG CHOO, IAN MACREADIE, SANGKOT MARZUKI, H. B. LUKINS, PHILLIP NAGLEY, AND ANTHONY W. LINNANE Department of Biochemistry, Monash University, Clayton, Victoria $168, Australia Received November 30, 1979 A temperature-conditional, respiratory-deficient mutant of Saccharomyces cerevisiae is described that results from mutation of mitochondrial DNA at a locus designated tsvl. Genetic mapping studies together with restriction endonuclease analysis of mitochondrial DNA from petite mutants retaining or deleted for this locus establish that the mutation is located in, or else is very closely linked to, the structural gene for the varl protein. The varl protein is a product of mitochondrial protein synthesis which in different strains exhibits variant molecular weights. Further evidence for the mutation occurring in the varl gene is provided by biochemical analysis of the mutant phenotype. Examination of mitochondrial translation products present in cells of the mutant strain grown at the permissive temperature (28°C) reveals a specific and significant reduction in the level of the varl protein. When grown at the restrictive temperature (36”C), the mutant strain shows an inhibition of the synthesis of varl protein, as well as inhibition of formation of subunit 1 of cytochrome oxidase and the proteolipid subunit of mitochondrial ATPase. These effects on mitochondrial translation prod- ucts can be correlated with a decreased mitochondrial protein synthetic activity in mutant cells grown at 36°C. These results are discussed in relation to the possible role of the varl protein. The mitochondrial translation system of the yeast Saccharomyces cerevisiae synthe- sizes at least eight protein components of the inner mitochondrial membrane (1). One of these products, designated varl, exists in several strain-dependent forms, (M, 40,000 to 47,000) (1). The structural gene of varl has been mapped on the mtDNA between the olil locus (coding for the proteolipid sub- unit of the mitochondrial ATPase (2-4)) and the 21 S rRNA gene, lying in a region close to the olil locus (5). The gene determining the varl protein is apparently allelic to the gene determining another mitochondrial product, var2, which is a 26,000 1M,peptide found in some wild-type yeast strains (1). is not yet established. Among the yeast strains which carry different forms of the varl protein there are no obvious physio- logical differences in terms of the capability to grow on nonfermentable energy sources(1). One approach to investigate the function of the varl protein would be to study mutants which show modified behavior as a result of a mutation affecting this polypep- tide. In this communication we describe the properties of a temperature-conditional mutant of S. cerevisiae which carries a mutation mapping in or very close to the varl gene, and which contains reduced amounts of the varl protein. The function of the varl protein is still unknown. Although this protein has been shown to be associated with the small sub- unit of the mitochondrial ribosome (6, 71,its function in mitochondrial protein synthesis MATERIALS AND METHODS Materials. The three protease inhibitors p-amino- benzamidine HCl, l -aminocaproic acid, and phenyl- methylsulfonyl fluoride were obtained from Sigma Chemical Company (St. Louis, MO.). SDS2was obtained f This paper is No. 54 in the series “Biogenesis 2 Abbreviations used: SDS, sodium dodecyl sulfate; of Mitochondria.” No. 53 is by Atchison et al. (12). kbp, kilobase pairs. OOo3-9861/80/090260-11$02.00/O Copyright 0 1980 by Academic Press, Inc. AI1 rights of reproduction in any form reserved. 260