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Submitochondrial Localization, Cell-Free Synthesis, And Proc. Nati Acad. Sci. USA Vol. 80, pp. 1270-1274, March 1983 Biochemistry Submitochondrial localization, cell-free synthesis, and mitochondrial import of 2-isopropylmalate synthase of yeast (leucine biosynthesis/synthase-deficient mutants/isoenzymes) D. MICHAEL HAMPSEY*t, ALFRED S. LEWINt, AND GUNTER B. KOHLHAW* *Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907; and *Department of Chemistry,. Indiana University, Bloomington, Indiana 47405 Communicated by H. E. Umbarger, December 13, 1982 ABSTRACT 2-Isopropylmalate synthase (EC 4.1.3.12) of yeast to be imported into the mitochondrion is made as a larger mo- is a mitochondrial enzyme. We now.provide evidence showing that lecular weight precursor, whether the import is post-transla- a large part of the 2-isopropylmalate synthase activity that is as- tional or cotranslational, and whether it requires metabolic en- sociated with the mitochondria is located in the mitochondrial ma- ergy. In thepresentpaperwe-address thesequestionswithrespect trix. In vitro translation of total yeast RNA followed by immu- to yeast 2-IPM synthase. noprecipitation with anti-2-isopropylmalate synthase antibody yields two polypeptides. The larger of these has an apparent molecular MATERIALS AND METHODS weight identical to that of purified 2-isopropylmalate synthase subunit (ca. 65,000). It is incorporated into isolated yeast mito- Strains. Two wild-type strains were used: S. cerevisiae D273- chondria with no detectable change in molecular weight. The im- 10B (a; ATCC 25657) and S288c (a SUC2 mal gal2 CUPI). Mu- port requires energy. The smaller polypeptide migrates to a po- tant strain HB190, kindly provided by H. E. Umbarger, is an sition corresponding to a molecular weight of 63,000-64,000. It is 2-IPM synthase-less leucine auxotroph derivedfrom S288c. Strain not taken up by mitochondria. Both polypeptides, which also can SK413 is a leucine-leaky mutant derived from S288c that also be obtained by immunoprecipitation of crude extracts, become la- has no detectable 2-IPM synthase activity (when assayed under beled when in vitro translation is performed in the presence of N- standard conditions). It was isolated by Y.-P. Hsu in the labo- formyl[35S]methionyl-tRNAf. Mutants with no detectable 2-iso- ratoryofone ofus (G.B.K.). Antibodywaspreparedagainsthighly propylmalate synthase activity are deficient in either one or both purified 2-IPM synthase from strain SK101, another derivative synthase-related polypeptides. These results are discussed in the of S288c (16). light of recent evidence for two 2-isopropylmalate synthase-en- Growth and Labeling of Cells. For the purpose of isolating coding genes in yeast. intact mitochondria, cells were grown on 1% yeast extract, 1% peptone, and 1% galactose to 250-500 Klett units. For other In both Saccharomyces cerevisiae and Neurospora crassa, a purposes, semisynthetic medium (17) containing 0.3% galactose number of enzymes involved in the biosynthesis of amino acids was used (unless otherwise specified). For preparing uniformly have been shown to be associated with the mitochondria. The labeled cells, this medium was supplemented with 30 ,uCi (1 Ci mitochondrial enzymes usually constitute a coherent portion of = 3.7 X 1010 Bq)-of carrier-free 5SO42- per ml; cells were har- a biosynthetic pathway. For example, in S. cerevisiae all of the vested when 90-95% of the label had been taken up. enzymes of the acetylglutamate cycle (catalyzing the early re- Isolation of Mitochondria. Mitochondria were isolated from actions in arginine biosynthesis) are found in mitochondria (1, spheroplasts according to published procedures (18, 19). 2). The same enzymes as well as ornithine transcarbamylase Cell-Free Protein Synthesis. Protein synthesis in nuclease- (ornithine carbamoyltransferase) and carbamoyl-phosphate syn- treatedrabbitreticulocyte lysates was done according to Pelham thetase A are mitochondrial in N. crassa (3-6). In both organ- and Jackson (20) except that 10 units of aprotinin per ml and 0.1 isms, the reactions ofthe common pathway leading to valine and mM phenylmethanesulfonyl fluoride were included in the isoleucine appear to take place largely in the mitochondria (7, translation mixture. High specific activity L-[LS]methionine was 8). Enzymes catalyzing the early reactions of lysine biosynthesis obtained from Amersham or New England Nuclear. For protein are associatedwith the mitochondriaofyeast (9, 10). Also in yeast, synthesis in the presence oflabeled andformylatedinitiator tRNA a high percentage of 2-isopropylmalate (2-IMP) synthase (EC (see below), 1 mM unlabeled L-methionine was included in the 4.1.3.12), the enzyme catalyzing the first committed step in leu- reaction mixture in order to prevent the labeling of proteins in cine biosynthesis, apparently is contained in the mitochondria internal methionine residues. When the labeled protein made (11). in vitro was to be used for import experiments (21), reactions It is generally agreed that the mitochondrial genome of yeast were stopped by chilling on ice and phenylmethanesulfonyl flu- andN. crassacodesforonlyasmallnumberofpolypeptideswhich, oride was added to 1 mM. Polysomes were then removed by with one exception, are involved in oxidative phosphorylation centrifugation at 140,000 x g for 45 min, and the lysates were (for review, see ref. 12). It is therefore very likely that all of the filtered over Sephadex G-25 columns equilibrated with "import mitochondrial enzymes mentioned above, including those whose buffer" (0.6 M mannitol/1S0 mM KCI/1 mM MgCI2/20 mM genes have not been mapped, are synthesized on cytoplasmic Hepes KOH, pH 7.4). ribosomes. One of the questions that arises in dealingwith such Acylation and Formylation of Yeast Initiator tRNA. Purified proteins (13-15) concerns their exact location within the mi- yeast initiator tRNA was the generous gift of Paul Sigler (Uni- tochondrion. Other pertinent questions are whether a protein Abbreviations: IPM, isopropylmalate; CCCP, carbonylcyanide m-chlo- Thepublication costs of this article were defrayed in partby page charge rophenylhydrazone; Mops, morpholinopropanesulfonic acid. payment. This article must therefore be hereby marked "advertise- tPresent address: Dept. of Radiation Biology and Biophysics, Univ. of ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Rochester Medical Center, Rochester, NY 14642. 1270 Downloaded by guest on September 26, 2021 Biochemistry: Hampsey et al. Proc. Natl. Acad. Sci. USA 80 (1983) 1271 versity of Chicago). It was acylated and formylated as described (22). The acylation and formylation extract was kindly donated by Joyce Heckman (Indiana University). Formylation of charged initiator tRNA by this extract was >80% complete. After charg- ing and formylation, yeast N-formyl[3S]methionyl-tRNAf was > 80 filtered over a Sephadex G-50 column and precipitated with ethanol before use in a rabbit reticulocyte lysate protein syn- 60 thesis system. _ t in Electrophoresis NaDodSO4/Polyacrylamide Gels. Poly- Z 40 - a-IPM SYNTHASE was acrylamide gel electrophoresis performed according to t CITRATE SYNTHASE Douglas et al (23). All samples were denatured with NaDodSO4 O and subjected to electrophoresis under disulfide-reducing con- 20 _- ditions. The concentration of acrylamide was 8% unless oth- erwise indicated. 0 15 30' 45 60 Enzyme Assays. 2-IPM synthase activity was determined by MINUTES measuring the amount of CoA produced within a timed incu- bation period by reaction with 5,5'-dithiobis (2-dinitrobenzoic FIG. 1. Effect of proteases on 2-IPM synthase (solid symbols) and acid) (24). Citrate synthase activity was determined by the same citrate synthase (open symbols) activities in intact and detergent-dis- procedure with oxaloacetate substituted for a-ketoisovalerate. rupted mitochondria. A and *, A mixture of 200 Al of a mitochondrial suspension [50 mg (wetweight)/ml] in 20 mM Mopsbuffer, pH 6.8, con- The unit of activity for both enzymes is defined as jmol of CoA taining 0.6 M mannitol, 1 mg of bovine serum albumin per ml, and 1 produced per hr. Cytochrome b2 was assayed for lactate de- mM EDTA with 0.2 mg each of trypsin and chymotrypsin (for 2-IPM hydrogenase activity (25). synthase) or 0.16 mg of proteinase K (for citrate synthase) in a final Miscellaneous Procedures. Published procedures were used volume of 240 Al was incubated at 3000. At the indicated times, 50-,ul for extraction of total yeast RNA (21), fluorography of dried slab portions were removed and added to 5 ul of a protease inhibitor so- and lution (phenylmethanesulfonyl fluoride, L-1-tosylamide-2-phenyleth- gels (26), methylation of 2-IPM synthase (27), immunopre- yl chloromethyl ketone, N-p-tosyl-L-lysine chloromethyl ketone, and cipitation of proteins (21). p-aminobenzamidine, each at 1 mM, in dimethyl sulfoxide). After 1 min, 2.5 ,ul of a 2% Triton X-100 solution was added to disrupt the mito- RESULTS chondria. Samples (10 ,ul) were then diluted 1:12.5 into assay medium for either 2-IPM synthase or citrate synthase. o and *, Same as above Determination of the Subcellular Location of 2-IPM Syn- except that proteases were omitted. o and e, Same as above except that detergent was added prior to proteases. thase. 2-IPM synthase previously was shown to be a particulate enzyme that sedimented with mitochondrial marker enzymes and behaved like citrate synthase in solubilization experiments activities were determined in whole mitochondria, a fraction (11). Still, the question remained as to whether 2-IPM synthase containing the intermembrane space, and the "mitoplasts. " is actually sequestered within the organelle or is only periph- The majority of both 2-IPM synthase and citrate synthase ac- erally associated with it. We approached this problem first by tivities remained with the mitoplasts; by contrast, the majority treating isolated intact mitochondria with proteases such that of the intermembrane space marker cytochrome b2 was found any protein associated with the outer surface of the organelle in the supernatant solution (Table 1). would be digested, whereas protein within the organelle would Cell-Free Synthesis and Mitochondrial Import of 2-IPM be protected.
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