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Molec. Biol. Rep. Vol. 4, 2:83~86 ALTERED MITOCHONDRIAL RIBOSOMES IN A COLD-SENSITIVE MUTANT OF SACCHAROMYCES CEREVISIAE Terence W. SPITHILL, K.J. ENGLISH, Phillip NAGLEY & Anthony W. LINNANE Department of Biochemistry, Monash University, Clayton, Victoria 3168, Australia (Received December 1, 19 77) Abstract A mutation at a new locus denoted tsrl which lies very close to the eryl locus and 21S rRNA gene in mitochondrial DNA of Saccharomyces cerevisiae, confers conditional respiratory deficiency on cells grown at low temperature, namely 18 ~ . Studies on mitochondria isolated from a strain carrying the mutated tsrl locus demonstrate that the rate of mitochondrial protein synthesis is cold-sensitive at t8 ~ The large subunit of the mitochondrial ribo- somes isolated from the mutant strain is unstable during extraction and the isolated ribosomes are shown to be defective in catalyzing the poly U- directed synthesis of polyphenylalanine. It is con- cluded that the tsrl locus is involved in the deter- mination of the properties of the large subunit of the mitochondrial ribosome. Introduction This laboratory has recently described the isolation and genetic characterization of a new cold-sensitive mutant of Saccharomyces cerevisiae (4,6). The conditional respiratory deficient phenotype (a markedly reduced growth rate at 18 ~ on non-fermen- table substrates) was shown to result from a mutation at a new locus in mitochondrial DNA (mtDNA), designated tsrl. Genetic and physical mapping techniques (4,6,10) were used to map the tsrl locus to within a short segment of mtDNA (approx. 4000 bp) which includes the cistron for the 21S rRNA component of the large ribosomal subunit and several antibiotic resistance loci (such as eryl and cap1) which affect ribosomal function (see ref. 10). However, mapping studies alone cannot assign the tsrl locus to a function involving the mitochon- drial ribosome. In this paper we demonstrate that a cold-sensitive strain 151K-2-2C carrying the tsrl mutation (4,6) in mtDNA contains mitochondrial ribosomes with altered properties. Both structural and functional defects are observed. Materials and methods The two strains of Saccharomyces cerevisiae used in this work are the standard respiratory competent strain L410 and the cold-sensitive mutant strain 151K-2-2C (for details of genetic derivation see ref. 6). Cultures of L410 or 151K-2-2C were grown at 28 ~ in the following medium: ethanol (1%), Difco yeast extract (1%), a salts mixture (13), and the necessary auxotrophic requirements: for L410, uracil and histidine (each 50/~g/ml); for 151 K-2-2C, adeni- ne, histidine and lysine (each 50 pg/ml). Large scale cultures were grown at 28 ~ under forced aeration in fermentors containing 11 litres of media. Cells were harvested in the logarithmic phase of growth (2-3 mg dry weight cells/ml). Mitochondria were prepared by the method of Cobon et al. (2) with the following modifications. Mitochondria were isolated in buffer A (0.6 M sorbitol, 10 mM EDTA, 6.6 mM Tris, pH 7.4). Crude mitochondria were collected at 12,000 x gay for 12 min, resuspended in buffer A and centrifuged at 1,000 x gay for 10 min to discard aggregated material. Mitochondria were again collected at 17,000 x gay for 12 rain and washed twice more in buffer A. Purified mitochondrial ribosomes were isolated from mitochondria using a high salt-detergent lysis procedure, followed by centrifugation through 1.2 M sucrose containing 0.2% Triton X-100 and 0.15 M NH4C1 as described elsewhere (5,11). The purified ribosomes were suspended in a buffer con- 83