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 otherwise 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 incubation 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. After a 60-min incubation with trypsin/chy- Synthase. Because 2-IPM synthase is apparently localized in the motrypsin, approximately 80% ofthe 2-IPM synthase remained mitochondrial matrix but is made in the cytosol, it must traverse unaffected, as defined by retention of enzymatic activity; mi- both mitochondrial membranes after its synthesis. Many mitochondria not exposed to proteases retained about 85% oftheir tochondrial proteins made in the cytoplasm are synthesized as activity during the same time period (Fig. 1). However, when the mitochondria were disrupted by detergent before incuba- Table 1. Distribution of enzymes between mitoplast and tion with proteases, all enzymatic activity was lost within 15 intermembrane space fractions min. Citrate synthase, a known mitochondrial matrix enzyme Mitochondria Intermembrane in a number oforganisms (28-30), was also assayed. In this case, before Mitoplast space proteinase K had to be substituted for trypsin/chymotrypsin Enzyme fractionation fraction fraction (free citrate synthase was resistant to digestion by trypsin/chymotrypsin, whereas free 2-IPM synthase was resistant to diges- 2-IPM synthase 5.2 3.5 ND tion by proteinase K), but the protection experiment yielded Citrate synthase 101 106 21 Cytochrome b2 3.0 0.6 2.1 essentially the same results as for 2-IPM synthase (Fig. 1). Further support for 2-IPM synthase being sequestered within The numbers represent enzyme units in 0.5 ml of a suspension of the mitochondria comes from the difficulty in assaying the en- mitochondria before fractionation and recovered after fractionation of zyme in isolated, intact organelles. In freshly isolated, un- the mitochondria into a mitoplast and an intermembrane space frac- treated mitochondria, only 6-27% of the 2-IPM synthase avail- tion. Freshly prepared mitochondria, 200-300 mg (wet weight) isolated from spheroplasts in the presence of 0.6 M mannitol, were care- able in Triton X-100-treated mitochondria was detected; similarly, fully suspended in 1 ml of 20 mM Mops buffer (pH 7.4) containing 0.1 only 14-37% of the citrate synthase was assayable in untreated M mannitol. Half of the suspension was treated with 2% Triton X-100 mitochondria. Because the outer membrane is not expected to and subjected to enzyme assays. The other half was allowed to stand be a barrier for the substrates or products of 2-IPM synthase and for 30 min at 0C and was then centrifuged for 10 min at 17,800 x g citrate synthase (31), it must be concluded that a large propor- (0WC). The supernatant solution (containing the intermembrane space tion of both enzymes is contained within the space bounded by enzymes) was decanted and the pellet (containing the mitoplasts) resuspended in 0.5 ml of 20 mM Mops buffer (pH 7.4) with 0.6 M man- the inner membrane. nitol. Both the resuspended pellet, after treatment with 2% Triton X- Evidence that 2-IPM synthase is not an intermembrane space 100 to lyse the mitoplasts, and the supernatant fraction were assayed enzyme was obtained from an experiment in which enzyme for the three enzymes indicated. ND, not detectable. Downloaded by guest on September 26, 2021 1272 Biochemistry: Hampsey et aL Proc. Natl. Acad. Sci. USA 80 (1983) precursors several thousand daltons larger than the "mature" densitometry (compare lanes 3 and 5). By contrast, practically proteins (i.e., those found inside the organelle). The import of all of the (nonimported) material found in the post-mitochon- proteins into mitochondria has been shown to be independent drial supernatant solution was protease-digested (compare lanes of protein synthesis (21) but to require an energized mitochon- 2 and 4). Fig. 2C shows that an energized mitochondrial mem- drial membrane (32, 33). Prior to our study of the mitochondrial brane is required for the import of 2-IPM synthase. The un- import of2-IPM synthase, 'S-labeled enzyme was synthesized coupler carbonylcyanide m-chlorophenylhydrazone (CCCP) in vivo and in vitro. Two radioactive polypeptides were pre- completely blocked the mitochondrial uptake of the enzyme cipitated with anti-2-IPM synthase antibody, both from whole- (compare lanes 1 and 2 of Fig. 2C with lanes 2 and 3 of Fig. 2B). cell extract of cells continuously labeled with 'SO42- and from The uncoupler may have this effect either by depleting mito- a rabbit reticulocyte lysate programed with total yeast RNA and chondrial pools of ATP (32) or, more likely, by eliminating the containing [3S]methionine (Fig. 2A). The larger of the two electrochemical gradient across the inner mitochondrial mem- polypeptides had the same apparent molecular weight as pu- brane (33). In contrast to CCCP, the presence of 2 mM o-phen- rified, mature 2-IPM synthase-about 65,000 (24). anthroline has no effect on the uptake of 2-IPM synthase by mi- The results depicted in lanes 2-5 of Fig. 2B demonstrate that, tochondria (34). This divalent metal chelator is known to inhibit of the two 2-IPM synthase-related polypeptides synthesized in a protease that processes polypeptide precursors destined for vitro, only the larger form was incorporated into the mitochon- mitochondrial import (12, 33). Its failure to affect the import of dria. Under the conditions chosen, about three-quarters of the 2-IPM synthase lends further support to the idea, derived from larger form actually appeared in the mitochondrial pellet (com- the previous results, that no higher molecular weight precursor pare lanes 2 and 3). The fact that, on the average, between 40% is required for the mitochondrial uptake of this protein. and 60% of this material had become resistant to trypsin and Translation in the Presence of N-Formyl[35S]Methionyl- chymotrypsin indicated that it had been imported into intact tRNAf. In order to find out whether the two polypeptides that organelles. In the experiment shown, 43% of the larger poly- are precipitated with anti-2-IPM synthase antibody are both peptide had become protease-resistant, as revealed by laser primary translation products, we labeled the protein synthesized in a rabbit reticulocyte lysate by using N-formyl[3S]- A B c methionyl-tRNAf ("initiator" tRNA). Radioactive tracer used in 1 2 3 4 1 2 3 4 6 1 2 this way can only be added to the NH2-terminal residue. Be- .. cause eukaryotic cells are apparently unable to remove N-for- mylmethionine from nascent protein (35), proteins recovered with label resulting from N-formyl[3S]methionyl-tRNAf could not have been subject to proteolytic processing of the NH2 ter- V."": minus. Both immunoreactive forms of 2-IPM synthase were la-

*:: 68 .* _s .: ::, _ _, _. _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.-:,c... 1 2 3

FIG. 2. (A) Comparison of 2-IPM synthase synthesized in vivo and in vitro with "mature" enzyme; fluorogram of an 8% NaDodSO4/polyacrylamide gel. Lanes: 1, commercially prepared 14C-labeled bovine serum albumin; 2, anti-2-IPM synthase antibody-reactive polypeptides from cells grown in the presence of 35SO42; 3, anti-2-IPM synthase antibody-reactive polypeptides from a cell-free translation mixture; 4, purified, [14C]methylated 2-IPM synthase. (B) Post-translational in- corporation of in vitro synthesized 2-IPM synthase into isolated mitochondria. After separation of the polysomes from the cell-free translation mixture and gel filtration through a Sephadex G-25 column, portions of the filtered lysate were incubated with isolated mitochondria (0.25 mg, wet weight) in the presence of 1 mM ATP, 5 mM phos- phoenolpyruvate, 6 units of pyruvate kinase, and 1 mM dithiothreitol in a final volume of 600 ,ul. After 20 min at 28°C, phenylmethanesulfonyl fluoride and N-p-tosyl-L-lysine chloromethyl ketone were added, to 1 mM each. This was followed by centrifugation to separate the mitochondriafrom the supernatant fraction. The pellet was washed once with 20 mM HepesKOH, pH 7.4/0.6 M mannitol and resuspended in 100 ,ul of the same buffer. NaDodSO4 was added to both fractions at a final concentration of 2.5% and the mixture was boiled for 3 min. 2-IPM synthase was then immunoprecipitated, electrophoresed, and FIG. 3. Cell-free synthesis of 2-IPM synthase in the presence of la- treatedforfluorography. Lanes: 1 and6, 2-IPM synthasefrom35SO42- beledinitiatortRNA. Rabbitreticulocytelysates were treated with nu- grown cells; 2 and 3, 2-IPM synthase from the post-mitochondrial su- clease (20) and programed with east RNA in the presence of either pernatant and mitochondrial fraction, respectively; 4 and 5, identical [1Slmethionine or ofN-formyl[ S]methionyl-tRNAf. Lanes: 1, 2-IPM to lanes 2 and 3, respectively, except that after mitochondrial uptake synthase immunoprecipitated fromcontinuously labeled yeast cells; 2, the suspension was treated with 0.6 ,ug of trypsin and 0.6 ug of chy- 2-IPM synthase immunoprecipitated from a reticulocyte lysateprotein motrypsin for 20 min at 0°C before addition of protease inhibitors. (C) synthesis system labeled with carrier-free [3S]methionine; 3, 2-IPM Inhibition of import of 2-IPM synthase by the uncoupler CCCP. Lanes: synthase immunoprecipitated from a reticulocyte lysate protein syn- 1 and 2, repetition of the experiment shown in lanes 2 and 3 inB except thesis system labeled in the presence of N-formyl(MSlmethionyl-tRNAf. that 10 pM CCCP was present during incubation of lysate with the Although all lanes are from the same gel, lanes 1 and 2 are shown as mitochondria. 3-day exposures ofthefluorogram whereaslane 3 is a 10-day exposure. Downloaded by guest on September 26, 2021 Biochemistry: Hampsey et al. Proc. Natl. Acad. Sci. USA 80 (1983) 1273 1 2 3 4 5 6 finding that only the larger polypeptide is taken up by the mitochondria. With mutant strain HB190, the largerband was still visible, but the smaller band was absent from the immunopre- cipitate of total cellular protein (lane 3). Much of the immunoprecipitable material representing the upper band was evi- dently still incorporated by the mitochondria (lane 4). Finally, with mutant strain SK413, no immunoactive polypeptides appeared at or near the position of the 2-IPM synthase standard (lanes 1 and 2). DISCUSSION In the last several years, a wealth of information has been generated concerning the mechanism by which proteins are imported into mitochondria (see refs. 12 and 37 for reviews re- garding the situation in yeast and N. crassa). Although the surface of yeast mitochondria contains cytoplasmic polysomes that are enriched in mRNA for mitochondrial proteins (37, 38), it appears that association of polysomes with mitochondria is not obligatory for protein import (37, 39). In fact, it has been shown that all cytoplasmically made mitochondrial proteins studied so far can be imported post-translationally. In this respect, 2-IPM synthase is no exception. However, unlike most other proteins destined for mitochondrial import, 2-IPM synthase is not synthesized as a larger precursor, within the ca. ±500-dalton limit ofresolution ofthe experiments (the precursors identified so far have extensions of 500-10,000 daltons). In fact, the only other published example of a matrix enzyme not made as a larger precursor is carbamoyl-phosphate synthetase from tadpole liver (40). It is not clear at this point by what mechanism 2-IPM synthase FIG. 4. Immunochemical detection of 2-IPM synthase in whole cell is "drawn" into or "trapped" within the matrix. However, it is and mitochondrial extracts from wild-type and 2-IPM synthase-defi- known that mature 2-IPM synthase exists as a dimer (24) and is cient yeast strains. NaDodSO4 extracts of whole cell homogenates or homogenates of isolated mitochondria were loaded onto an 8% poly- a zinc metalloenzyme (41). Thus, the translocation of the en- acrylamide gel. After electrophoresis, proteins were transferred to a zyme could be facilitated either by conformational changes in- nitrocellulose sheet as described (36). The immobilized bands were then volving the quaternary structure or by addition of the metal in- allowed to react with antiserum raised against 2-IPM synthase. The side the matrix. Both conformational change (ADP/ATP carrier) presence of rabbit antiserum bound to the nitrocellulose sheet was de- and conversion of apoenzyme to holoenzyme (cytochrome c) have tected by using horseradish peroxidase-conjugated goat antiserum di- been discussed as possible driving forces for vectorial transport rected against rabbit IgG. The entire procedure was as described (36). Lanes: 1, whole cell extract from strain SK413 (synthase-deficient); 2, (37, 42). It is also possible that translocation is "spontaneous" mitochondrial extract from strain SK413; 3, whole cell extract from along the lines of the membrane-triggered folding model dis- strain HB190 (synthase-deficient); 4, mitochondrial extract from strain cussed by Wickner (43) for proteins that become integral parts HB190; 5, whole cell extract from strain S288ca (wild type, parental of membranes. strain); 6, mitochondrial extract from strain S288ca. The position of 2- A surprising result of the present study was the finding that IPM synthase on the nitrocellulose sheet was determined by using a immunoprecipitation with anti-2-IPM synthase antibody con- 35S-labeled standard not treated with antiserum and is indicated by an arrow. The uppermost band is from methyl green used to monitortrans- sistently elicited the appearance of two polypeptides. Several fer of proteins possibilities may be considered to explain the presence of a sec- to the nitrocellulose sheet. ond immunoreactive polypeptide. (i) It could be an artifact of the immunoprecipitation pro- beled when the translation was carried out in the presence of cedure. Although this possibility cannot be rigorously excluded, N-formyl['S]methionyl-tRNAf (Fig. 3). it must be reiterated that the antibody was raised against 2-IPM Immunoprecipitation of Protein from Mutants Deficient in synthase of very high purity (34). It therefore is unlikely that 2-IPM Synthase. In an attempt to correlate enzyme activity with significant amounts of a second antibody, unrelated to 2-IPM the pattern seen upon electrophoresis of polypeptides precip- synthase, were generated. Moreover, the facts that neither of itated with anti-2-IPM synthase antibody, two mutants were two 2-IPM synthase-deficient mutants contains detectable analyzed that showed no detectable 2-IPM synthase activity when amounts of the faster moving immunoreactive band and that assayed by the standard assay procedure (i.e., the level of active one of these mutants contains neither band indicate that both 2-IPM synthase in the mutants was <10% of the wild-type level). polypeptides are related to functional 2-IPM synthase. The appearance of a relatively large number of bands in this ex- (ii) The smaller polypeptide could be a product of the larger periment (Fig. 4) was probably caused by the great sensitivity one. Because both bands were labeled in an in vitro translation of the immunoreplication technique used. It is obvious, never- experiment performed in the presence of N-formyl[3S]methio- theless, that major differences occur only at or near the position nyl-tRNAf, the smaller polypeptide could not have been gen- of the 2-IPM synthase standard. When total cellular protein of erated from the larger one by proteolysis at the NH2 terminus. a wild-type strain was subjected to immunoprecipitation (lane It could have been generated by proteolysis at the COOH ter- 5), two bands appeared at this position with relative densities minus, but this also is unlikely in view of preliminary results of similar to those seen in previous whole cell experiments (see a pulse-chase experiment which showed that there was no sig- Fig. 2). Only the upper band appeared when mitochondrial pro- nificant change in the relative densities of the two bands during tein was immunoprecipitated (lane 6), confirming the previous 60 min of chase. Downloaded by guest on September 26, 2021 1274 Biochemistry: Hampsey et al. Proc. Nad Acad. Sci. USA 80 (1983) (iii) The larger polypeptide band could be a product of the 8. Ryan, E. D. & Kohihaw, G. B. (1974)J. BacterioL 120, 631-637. smaller one. This would probably require a rather specific post- 9. Betterton, H., Fjellstedt, T., Matsuda, M., Ogur, M. & Tate, R. translational covalent modification, an event unlikely to happen (1968) Biochim. Biophys. Acta. 170, 459-461. 10. Tracy, J. W. (1976) Dissertation (Purdue Univ., West Lafayette, in our in vitro protein-synthesizing system that uses rabbit re- IN). ticulocyte lysate and does not contain any yeast component other 11. Ryan, E. D., Tracy, J. W. & Kohlhaw, G. B. (1973)J. Bacteriol. than RNA. The results of the pulse-chase experiment men- 116, 222-225. tioned above also argue against this possibility. 12. Schatz, G. (1981) in Mitochondria and Microsomes, eds. Lee, C. (iv) The two bands could represent the products of two genes P., Schatz, G. & Dallner, G. (Addison-Wesley, Reading, MA), pp. coding for 2-IPM synthase. This possibility must be given se- 45-66. 13. Schatz, G. (1979) FEBS Lett. 103, 203-211. rious consideration because of mounting evidence that there are 14. Ades, I. Z. & Butow, R. A. (1980)J. BioL Chem. 255, 9925-9935. indeed 2-IPM synthase isoenzymes. For example, tetrad anal- 15. Harmey, M. A., Hallermayer, G., Korb, H. & Neupert, W. (1977) ysis of crosses between the synthase-less strain HBL90 and LEU+ Eur. J. Biochem. 81, 533-544. strains has resulted in segregation patterns that are consistent 16. Hampsey, D. M. & Kohlhaw, G. B. (1981)j.BioL Chem. 256, 3791- with the existence of two loosely linked genes, each of which 3796. is capable of providing adequate, albeit not optimal, 2-IPM syn- 17. Djavadi-Ohaniance, L., Rudin, Y. & Schatz, G. (1978)j BioL Chem. 253, 4402-4407. thase function (unpublished data). 18. Woodrow, G. & Schatz, G. (1979)J. BioL Chem. 254, 6088-6093. There is also other, more circumstantial, evidence such as the 19. Daum, G., Bohni, P. C. & Schatz, G. (1982)J. BioL Chem. 257, observation that 2-IPM synthase from glucose-grown cells shows 13028-13033. a sensitivity to leucine that was apparently different from that 20. Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, 247- seen with enzyme from cells grown on acetate as the sole carbon 256. source and the well-known encountered in at- 21. Maccechini, M.-L., Rudin, Y., Blobel, G. & Schatz, G. (1979) Proc. (44), difficulties NatL Acad. Sci. USA 76, 343-347. tempts to isolate synthase-negative mutants. 22. Lewin, A. S., Gregor, I., Mason, T. L., Nelson, N. & Schatz, G. It is of interest that the smaller of the immunoprecipitable (1980) Proc. Nati Acad. Sci. USA 77, 3998-4002. polypeptides was always present in lesser amounts (compared 23. Douglas, M., Finkelstein, D. & Butow, R. A. (1979) Methods En- to the larger polypeptide) when experiments were performed zymot 56, 58-66. with whole cells and that the two polypeptides were found in 24. Tracy, J. W. & Kohlhaw, G. B. (1977)J. BioL Chem. 252, 4085- about equal amounts in translation experiments performed in 4091. 25. Symons, R. H. & Burgoyne, L. A. (1966) Methods EnzymoL 9, vitro. We cannot offer a satisfactory explanation for these ob- 314-321. servations, but it is possible that, in vivo, either the translation 26. Chamberlain, J. P. (1979) AnaL Biochem. 98, 132-135. of the mRNA for the smaller polypeptide is less efficient or the 27. Rice, R. H. & Means, G. E. (1971) J. BioL Chem. 246, 831-832. polypeptide itself is less stable. 28. Brdiczka, D., Pette, D., Brunner, G. & Miller, J. (1968) Eur. J. In articles published after completion of the work reported Biochem. 5, 294-304. also evidence 29. Srere, P. A., Pavelka, S. & Das, N. (1971) Biochem. Biophys. Res. here, Schatz and co-workers presented showing Commun. 44, 717-723. that 2-IPM synthase of yeast is located in the mitochondrial ma- 30. Mukkerjee, A. & Srere, P. A. (1976)J. BioL Chem. 251, 1476-1480. trix and is not made as a larger molecular weight precursor (33, 31. Zalman, L. S., Nikaido, H. & Kagawa, Y. (1980)J. Biol Chem. 255, 39, 45). However, those authors did not observe the second, 1771-1774. faster moving band of immunoprecipitable material seen in our 32. Nelson, N. & Schatz, G. (1979) Proc. Nati Acad. Sci. USA 76, 4365- experiments. The major difference between their experiments 4369. 2-IPM 33. Gasser, S. M., Daum, G. & Schatz, G. (1982)J BioL Chem. 257, and ours appears to have been the antibody against syn- 13034-13041. thase. The antiserum used by them and the one used in our ex- 34. Hampsey, D. M. (1982) Dissertation (Purdue Univ., West Lafa- periments were made against different preparations of highly yette, IN). purified 2-IPM synthase. Resolution of this apparent discrep- 35. Palmiter, R. D., Gagnon, J., Ericsson, L. H. & Walsh, K. A. (1977) ancy will have to await the results of further experimentation. J. Biol Chem. 252, 6386-6393. 36. Towbin, H., Staehelin, T. & Gorom, J. (1979) Proc. Nati Acad. We are indebted to Dr. Gottfried Schatz (Biozentrum Basel) for stim- Sci. USA 76, 4350-4354. ulating and helpful discussions and for providing us with a procedure for 37. Neupert, W. & Schatz, G. (1981) Trends Biochem. Sci. 6, 1-4. mitochondrial subfractionation prior to publication of it. This work was 38. Ades, I. Z. & Butow, R. A. (1980)J. BioL Chem. 255, 9918-9924. supported by Public Health Service Grants GM15102 (G.B.K.) and 39. Suissa, M. & Schatz, G. (1982) J. BioL Chem. 257, 13048-13055. 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