RICHTER
Production of Mitochondrial Peptide-Chain Elongation Factors in Yeast Deficient in Mitochondrial Deoxyribonucleic Acid* Dietmar Richter
ABSTRACT : Mutants of the yeast Saccharomyces cerecisiae lacking mitochondrial DNA contain the complete set of mitochondrial elongation factors ; these enzymes are identical
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n preceding studies (Richter and Lipmann, 1970a; Perani et ai., 1971) it was shown that a set of elongation factors T and G could be isolated from yeast mitochondria which complemented ribosomes from mitochondria and Escherichia coli but not those from the cytoplasm. These factors could then be identified relatively easily in chromatograms of whole cell homogenates, and the mitochondrial T and G factors could thus be separated from their cytoplasmic counterparts. The preparation of mitochondrial factors without isolating the mitochondria has now been improved and rather highly purified T and G factors have been obtained. In order to determine if these mitochondrial factors were mapped on mitochondrial DNA, advantage was taken of the availability of yeast mutants free of this DNA. Mutants of the yeast Saccharomyces cereuisiue, which are deficient in mitochondrial DNA, may be produced by treatment with ethidium bromide (Slonimski et al., 1968; Perlman and Mahler, 1971). Marmur and his colleagues showed such mutants to have no detectable mitochondrial D N A (Goldring et ul., 1970); they were therefore well suited for our purpose. Two of these mutants were used in the following experiments. The results t o be reported show that the content of elongation factors in these mutants is identical with that of the wild-type yeast. Experimental Section The following strains from S. cerecisiue were used: the wild-type strain 18A, and the “petite” mutants 11-1-40 and 111-1-7; all three strains were obtained from Dr. Marmur, Albert-Einstein College of Medicine. The two “petite” mutants lack mitochondrial D N A (J. Marmur. personal communication). Growth Conditions. Strain 18A was grown in a yeast extract-peptone-salt medium containing, per 1.: 5 g of yeast extract, 10 g of peptone, 6 g of (NHJ2HPO1, 2 g of MgSO,, 9 g of KCL, and 33 ml of a 6 0 x lactate syrup. The medium was adjusted to p H 4.5 with HCL. A 10-1. carboy was inocculated with 300 ml of an overnight culture containing 7.0 A a j o units/ml. The cells were grown at 30” in a New Brunswick fermentor under aeration ( 2 1. of airlmin) with stirring (800 rpm). Growth was stopped a t a turbidity of 8.0 as measured from the 450-nm absorbance; the culture was quickly cooled
* From The Rockefeller University, New York, New York 10021. Receired JUIJ)19, 1971. This study was supported by a grant to Fritz Liprnann from the U. S. Public Health Service (GM-13972), and by a fellowship to the author from Deutsche Forschungsgemeinschaft.
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in their functional, protein chemical, and immunological properties with their counterparts in the parent strain.
and centrifuged in a continuous-flow rotor. The “petite” mutants were grown under similar conditions, except that the lactate was replaced by 1.5% glucose. The yield for all three strains was about 7-10 g of cells (wet weight) per 1. of medium. Preparafion of Miinchondrial Elongation Fortors. The same procedure for isolating the T and G factors may be applied to the wild-type strain 18A and the mutants 11-1-40 and III1-7. Mitochondrial T and G factor activity, respectively. were assayed with 50 pg of E. coli ribosomes supplemented with complementary bacterial factor (Richter and Lipmann, 1970a). The progress of purification is described in Table I for the wild-type strain, and in Table I1 results are compared with those obtained with the mutants. The total amount of T and G enzyme units was calculated for each purification step, and then expressed per gram of protein originally present in the 105,000g supernatant fraction. The data indicate that the mutant contains slightly less enzyme units per gram of supernatant protein than the wild-type strain. Yeast cells (200 g wet weight) were mixed with 400 ml of buffer 1 (20 inhi Tris-HCI (pH 7.4)-1 mbi DTT‘) and were homogenized in a Manton-Gaulin homogenizer as described (Richter and Lipmann, 1970a). The homogenate was centrifuged at 5000g for 10 min, and the cell pellet was reextracted with 300 ml of bufkr 1 and centrifuged as above. The supernatant fractions were combined and centrifuged twice, first at 18,000g for 20 min, then at 78,000g for 2 hr. This fraction is referred t o as S-100 (step 1 of Table I). The S-100 fraction (1040 ml) was adjusted to pH 6.8, precipitated with 70 g of ammonium sulfate/100 ml of solution, and centrifuged at 1 8 , 0 0 0 ~for 1 hr. The protein pellet was reextracted three times with 150 ml of 25%, then three times with 22 %, and finally three times with 18 ammonium sulfate solutions (wiw); all solutions contained 1 mM DTT; the pH was adjusted t o 6.8. The supernatant fractions of each step were combined and reprecipitated with 20 g of ammonium sulfateil00 ml of solution. The 2 5 % fraction contained no mitochondrial elongation factors and was discarded. The 22
