Proc. NatL Acad. Sci. USA Vol. 79, pp. 267-271, January 1982 Biochemistry Imported mitochondrial proteins cytochrome b2 and cytochrome cl are processed in two steps (mitochondrial biogenesis/protein import/proteolytic processing/in vitro protein synthesis/vectorial processing) SUSAN M. GASSER, AKIRA OHASHI, GUNTHER DAUM, PETER C. BOHNI, JANE GIBSON, GRAEME A. REID, TAKASHILYONETANI, AND GOTTFRIED SCHATZ Department of Biochemistry, Biocenter, University of Basel, CH-4056, Basel, Switzerland Communicated by Tadeus Reichstein, September 14, 1981 ABSTRACT Cytochrome b2 of yeast is located in the space 1 2 3 4 5 6 between the inner and outer mitochondrial membranes whereas cytochrome cl is bound to the outer face ofthe mitochondrial inner membrane. Both proteins are made outside the mitochondria as larger precursors that are processed to their mature forms in two pre b, steps. In the first step, at least a segment of the precursor poly- cyt b2 - peptide chain penetrates into the mitochondrial matrix and is cleaved to an intermediate form by a matrix-localized soluble pro- tease. This step requires an electrochemical gradient across the mitochondrial inner membrane. In the second step, the inter- mediate form is converted to the mature form. For cytochrome cl, this second step requires heme. Import of these two cyto- chromes thus involves a "detour" across the inner membrane. Most mitochondrial proteins are synthesized outside the mi- are than the cor- tochondria as precursors that usually larger FIG. 1. Import of cytochrome b2 into isolated yeast mitochondria responding mature polypeptides found inside the mitochondria is dependent on ATP. Total yeast mRNA was translated in a reticu- (1, 2). Very little is known about how these precursors are trans- locyte lysate in the presence of [35S]methionine (13), centrifuged for ported into the mitochondria. A promising experimental system 40 min at 140,000 x g to remove ribosomes, and filtered through a for studying this question is the uptake ofradiolabeled in vitro- Sephadex G-25 column equilibrated with 0.15 M KCI/20 mM Hepes- synthesized precursors by isolated mitochondria. In vitro up- KOH, pH 7.4. Aliquots (150 AD) of the filtered lysate were incubated take does not require protein synthesis and, with larger pre- with isolated yeast mitochondria (200 ,ug of protein; total vol, 0.4 ml) for 30 min at 280C. Samples in lanes 1 and 2 also contained 1 mM ATP/ cursors, is accompanied by conversion of the precursor to its 1 mM MgCl2/5 mM phosphoenolpyruvate and 4 units of pyruvate ki- mature size ("vectorial processing"; ref. 1). We have isolated nase. Samples in lanes 1, 2, 5, and 6 were subsequently treated for 30 a soluble chelator-sensitive protease from the yeast mitochon- min at 00C with 150 pg of trypsin/ml and then with 3 mg of soybean drial matrix that processes precursors of those proteins trans- trypsin inhibitor and 1 mM tosyl-L-lysyl-chlormethyl ketone. Mito- ported partly or completely across the inner membrane (3). chondria were then sedimented (10 min at 10,000 x g) and pellets these to the corresponding mature (lanes 1, 3, and 5) and supernatants (lanes 2,4, and 6) were dissociated Processing of precursors ir>3% NaDodSO4and subjectedto immunoprecipitation (13) with anti- polypeptides appears to occur in a single step. serum against cytochrome b2. The immunoprecipitated samples were Here we report that cytochrome b2, a soluble enzyme ofthe analyzed by NaDodSO/10% polyacrylamide gel electrophoresis and mitochondrial intermembrane space, is processed to its mature fluorography. A photograph of the fluorogram is shown. pre b2, Cy- size in two steps. Surprisingly, the first ofthese steps occurs in tochrome b2 precursor; cyt b2, mature cytochrome b2. the matrix space. Cytochrome cl, an inner membrane enzyme the space, is processed by a protruding into intermembrane cytochrome cl) or 10% (for cytochrome b2) gel slabs (8). Pub- similar two-step mechanism. lished methods were used for assaying protein (9), fumarase (10), and L-lactate-ferricyanide reductase (11); for fluorography METHODS ofdried gel slabs (12); and for isolating crude yeast mRNA (13), Growth and Labeling of Cells. Wild-type Saccharomyces spheroplasts (7), yeast mitochondria (14), soluble processing cerevisiae strain D273-10B (ATCC 25657) was grown on semi- protease from a yeast mitochondrial matrix (3), and yeast cy- synthetic medium (4) containing 0.3% galactose (unless stated tochrome cl (15). The isolation of subfractions from yeast mi- otherwise). The heme-deficient mutant GL-1 (5) was grown on tochondria will be described in detail elsewhere but details are semisynthetic medium containing 0.26% Tween 80, 12 ppm of available on request. ergosterol, and 2% glucose. Uniform labeling of cells with Immunological Methods. A monoclonal antibody against 3So2- or [3S]methionine (4) and pulse labeling of intact cells subunit III ofyeast cytochrome oxidase was raised by injecting (6) or spheroplasts (7) have been described. holocytochrome c oxidase into BALB/c mice and fusing spleen Analytical and Preparative Methods. NaDodSO4poly- lymphocytes with X63-Ag8 myeloma cells (16). Published meth- acrylamide gel electrophoresis was carried out on 12.5% (for ods were used for raising antisera in rabbits (17), for immuno- precipitation ofcell fractions dissociated in NaDodSO4 (13), and The publication costs ofthis article were defrayed in part by page charge for immune replication (18). payment. This article must therefore be hereby marked "advertise- Cell-Free Translation. Rabbit reticulocyte lysates were ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. mied with the necessary cofactors (19), frozen in liquid N2, and 267 Downloaded by guest on September 27, 2021 268 Biochemistry: Gasser et al. Proc. Natl. Acad. Sci. USA 79 (1982) Table 1. Import of cytochrome b2 into isolated mitochondria 1 2 3 4 Mitochondria Supernatant Trypsin absent 50 42* Trypsin present 47 <1 _ _pre b. Results are relative to amount of precursor immunoprecipitated cvt b.) from lysate before incubation (defined as 100%). Isolated yeast mito- chondria were allowed to import in vitro-synthesized precursor of cy- tochrome b2 in the presence of ATP as described in Fig. 1. To ensure equal efficiency of immunoprecipitation from the different samples, their contents of unlabeled cytochrome b2 were equalized by adding 200 ,g of unlabeled yeast mitochondria to each supernatant and to an aliquot of the labeled reticulocyte lysate that served as a 100% control. The samples were then dissociated with NaDodSO4 and treated as in Fig. 1. The fluorograph was quantitated with a Bausch and Lomb den- sitometer. Results refer to mature cytochrome b2 except where indi- cated otherwise. FIG. 2. ATP-dependent import of cytochrome b2 into isolated mi- * 31% precursor/11% mature. tochondria is blocked by carboxyatractylate. The experiment was sim- ilar to that described in Fig. 1 except that all samples contained the ATP mixture; trypsin treatment was omitted; and during incubation stored at -80'C without nuclease treatment. Theywere labeled ofthe mitochondria withlysate, 20 pg ofcarboxyatractylate was added with [3S]methionine [=100 Ci/mmol; 10 mCi/ml (1 Ci = 3.7 to the samples in lanes 3 and 4. Lanes: 1 and 3, pellet; 2 and 4, super- x 1010 becquerels); prepared according to ref. 20 from natant, pre b2, Cytochrome b2 precursor; cyt b2, mature cytochrome b2. 35SO2-] in the presence of 10-15 OD260 units of crude yeast mRNA per ml for 60 min at 29°C and treated further as outlined duced in vitro by incubating isolated yeast mitochondria with in Fig. 1. in vitro-synthesized cytochrome b2 precursor in the presence Materials. Yeast cytochrome b2 was a gift of F. Lederer and ofATP. Under these conditions, 50% ofthe labeled cytochrome F. S. Matthews. A rabbit antiserum against yeast isopropyl- b2 precursor is taken up by the mitochondria and converted to malate synthase was donated by P. Roeder and G. Kohlhaw. mature cytochrome b2 (Fig. 1), which is almost completely re- All other chemicals were purchased. sistant to externally added trypsin (Table 1). In the absence of ATP, the precursor is neither taken up nor processed (Fig. 1). RESULTS Vectorial processing of precursor polypeptides by isolated mi- Cytochrome b2 (L-lactate-cytochrome c reductase) of yeast is tochondria is thus energy dependent. This confirms the earlier coded in the nucleus, synthesized outside the mitochondria, observation that vectorial processing ofmitochondrial precursor and imported into the organelles (21). The mature enzyme is polypeptides in yeast spheroplasts requires energy (7). a soluble component of the mitochondrial intermembrane ATP-dependent import and processing ofcytochrome b2 are space. This location is documented by the following observa- blocked ifentry of the added ATP into the mitochondrial matrix tions (unpublished data): (i) on subfractionating yeast mito- is prevented by carboxyatractylate (Fig. 2). chondria, cytochrome b2 cofractionates with adenylate kinase Addition ofoligomycin (which blocks mitochondrial ATPase, (an intermembrane space marker), but not with fumarase, acon- ref. 22) or ofvalinomycin/K+ (which results in a collapse ofthe itase, or citrate synthase (three matrix markers); (ii) in intact membrane potential, ref. 22) also completely inhibits the im- mitochondria, the enzyme readily reduces added ferricyanide port of cytochrome b2 into mitochondria. However, if mito- (which can penetrate the outer but not the inner membrane) chondria are energized with a respiratory substrate such as suc- but fails to reduce added cytochrome c (which cannot penetrate cinate, instead ofwith ATP, oligomycin does not inhibit import either of the two membranes); and (iii) an antiserum against (not shown). Import of precursors into mitochondria thus re- cytochrome b2 inhibits the mitochondria-bound enzyme only quires an electrochemical gradient across the mitochondrial in- if the outer membrane is disrupted. ner membrane. The mitochondrial outer membrane cannot sus- Import of cytochrome b2 into mitochondria can be repro- tain such a gradient because it permits diffusion of molecules Table 2.