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• W1980258314 abstract "MAS17 (MAS22) is an essential component of the import receptor complex in the yeast mitochondrial outer membrane. MAS17 consists of three distinct domains: the N-terminal cytosolic domain, the internal membrane-spanning domain, and the C-terminal intermembrane space domain. In the present study, we examined the roles of the C-terminal domain of MAS17, which is rich in acidic amino acids, in protein import into mitochondria both in vivo and in vitro. Cells expressing MAS17Δ120-152, a mutant MAS17 lacking the C-terminal acidic domain, could grow as fast as those expressing wild-type MAS17, while cells expressing MAS17Δ97-152, a mutant MAS17 lacking both the intermembrane space and the membrane-spanning domains, stopped growing as soon as wild-type MAS17 was depleted. MAS17Δ120-152 was correctly integrated into the mitochondrial outer membrane like wild-type MAS17. Mitochondria containing MAS17Δ120-152 instead of wild-type MAS17 could import both authentic and artificial mitochondrial precursor proteins nearly as efficiently as wild-type mitochondria in vitro. These results suggest that the C-terminal intermembrane space domain of MAS17 is not essential for targeting or functions of MAS17. MAS17 (MAS22) is an essential component of the import receptor complex in the yeast mitochondrial outer membrane. MAS17 consists of three distinct domains: the N-terminal cytosolic domain, the internal membrane-spanning domain, and the C-terminal intermembrane space domain. In the present study, we examined the roles of the C-terminal domain of MAS17, which is rich in acidic amino acids, in protein import into mitochondria both in vivo and in vitro. Cells expressing MAS17Δ120-152, a mutant MAS17 lacking the C-terminal acidic domain, could grow as fast as those expressing wild-type MAS17, while cells expressing MAS17Δ97-152, a mutant MAS17 lacking both the intermembrane space and the membrane-spanning domains, stopped growing as soon as wild-type MAS17 was depleted. MAS17Δ120-152 was correctly integrated into the mitochondrial outer membrane like wild-type MAS17. Mitochondria containing MAS17Δ120-152 instead of wild-type MAS17 could import both authentic and artificial mitochondrial precursor proteins nearly as efficiently as wild-type mitochondria in vitro. These results suggest that the C-terminal intermembrane space domain of MAS17 is not essential for targeting or functions of MAS17. INTRODUCTIONNuclear-encoded mitochondrial precursor proteins are imported from the cytosol into mitochondria; they are recognized by cytosolic factors and/or mitochondrial receptor proteins and are subsequently translocated across the outer and inner mitochondrial membranes(1.Segui-Real B. Stuart R.A. Neupert W. FEBS Lett. 1992; 313: 2-7Crossref PubMed Scopus (44) Google Scholar, 2.Schatz G. Protein Sci. 1993; 2: 141-146Crossref PubMed Scopus (72) Google Scholar). Translocation across the mitochondrial membranes requires coordinated functions of two separate machineries in the outer and inner mitochondrial membranes; they most likely interact transiently to allow the movement of precursor proteins into the matrix(3.Horst M. Hilfiker-Rothenfluh S. Oppliger W. Schatz G. EMBO J. 1995; 14: 2293-2297Crossref PubMed Scopus (83) Google Scholar, 4.Berthold J. Bauer M.F. Schneider H.-C. Klaus C. Dietmeier K. Neupert W. Brunner M. Cell. 1995; 81: 1085-1093Abstract Full Text PDF PubMed Scopus (154) Google Scholar).MAS20 (5.Hines V. Brandt A. Griffiths G. Horstmann H. Brütsch H. Schatz G. EMBO J. 1990; 9: 3191-3200Crossref PubMed Scopus (203) Google Scholar) and MAS70 (6.Ramage L. Junne T. Hahne K. Lithgow T. Schatz G. EMBO J. 1993; 12: 4115-4123Crossref PubMed Scopus (180) Google Scholar) in Saccharomyces cerevisiae and MOM19 (7.Söllner T. Griffiths G. Pfaller R. Pfanner N. Neupert W. Cell. 1989; 59: 1061-1070Abstract Full Text PDF PubMed Scopus (247) Google Scholar) and MOM72 (8.Söllner T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 1990; 62: 107-115Abstract Full Text PDF PubMed Scopus (216) Google Scholar) in Neurospora crassa serve as receptor proteins and are responsible for the initial binding of mitochondrial precursor proteins to the mitochondrial surface. Yeast MAS37 may form a heterodimer complex with MAS70 and facilitate the receptor function of MAS70(9.Gratzer S. Lithgow T. Bauer R.E. Lamping E. Paltauf F. Kohlwein S.D. Haucke V. Junne T. Schatz G. Horst M. J. Cell Biol. 1995; 129: 25-34Crossref PubMed Scopus (152) Google Scholar). ISP42 in yeast (10.Baker K. Schaniel A. Vestweber D. Schatz G. Nature. 1990; 348: 605-609Crossref PubMed Scopus (195) Google Scholar) and MOM38 in N. crassa(11.Kiebler M. Pfaller R. Söllner T. Griffiths G. Horstmann H. Pfanner N. Neupert W. Nature. 1990; 348: 610-616Crossref PubMed Scopus (192) Google Scholar) likely mediate the step of subsequent protein translocation across the outer membrane. ISP42/MOM38 was found to be in contact with precursor proteins arrested in transit across the mitochondrial membranes(12.Vestweber D. Brunner J. Baker A. Schatz G. Nature. 1989; 341: 205-209Crossref PubMed Scopus (192) Google Scholar, 13.Söllner T. Rassow J. Wiedmann M. Schlossmann J. Keil P. Neupert W. Pfanner N. Nature. 1992; 355: 84-87Crossref PubMed Scopus (129) Google Scholar), and antibodies against ISP42 blocked protein import into mitochondria(12.Vestweber D. Brunner J. Baker A. Schatz G. Nature. 1989; 341: 205-209Crossref PubMed Scopus (192) Google Scholar).Another outer membrane protein MOM22 in N. crassa appears to function downstream of the receptors because antibodies against MOM22 inhibited import of mitochondrial precursor proteins into mitochondria but not their binding to receptors in vitro(14.Kiebler M. Keil P. Schneider H. van der Klei I.J. Pfanner N. Neupert W. Cell. 1993; 74: 483-492Abstract Full Text PDF PubMed Scopus (154) Google Scholar). MAS17 (MAS22), the yeast equivalent of N. crassa MOM22, has been identified recently(15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar, 16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar, 17.Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 2289-3382Crossref Scopus (112) Google Scholar). The MAS17 gene is essential (15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar, 16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar, 17.Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 2289-3382Crossref Scopus (112) Google Scholar) and depletion of functional MAS17 results in accumulation of the precursor form of a mitochondrial protein(16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar). MAS17 consists of three distinct domains: the N-terminal domain that is highly acidic and faces the cytosol, the internal hydrophobic domain that is integrated into the outer membrane, and the C-terminal domain that contains several acidic residues and faces the intermembrane space. The cytosolic acidic domain may well provide a binding site for positively charged presequences of mitochondrial precursor proteins.A key question concerning the protein import into mitochondria is what drives the vectorial movement of precursor proteins across the two membranes. In the case of protein translocation across the inner mitochondrial membrane, the membrane potential across the inner membrane triggers the movement of a positively charged presequence across the inner membrane probably by electrophoretic effects(18.Roise D. Horvath S.J. Tomich J.M. Richards J.H. Schatz G. EMBO J. 1986; 5: 1327-1334Crossref PubMed Scopus (308) Google Scholar, 19.Martin J. Mahlke K. Pfanner N. J. Biol. Chem. 1991; 266: 18051-18057Abstract Full Text PDF PubMed Google Scholar). Then mitochondrial hsp70 1The abbreviations used are: hsp7070-kDa heat shock proteinpF1βthe precursor of the F1-ATPase β subunitpCOXIV-DHFRthe presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductasepSu9-DHFRthe presequence of the F0-ATPase subunit 9 fused to mouse dihydrofolate reductasePAGEpolyacrylamide gel electrophoresis. likely drives the movement of the rest of the precursor polypeptide chain at the expense of ATP hydrolysis in the matrix(20.Schneider H.-C. Berthold J. Bauer M.F. Dietmeier K. Guiard B. Bruner M. Neupert W. Nature. 1994; 371: 768-774Crossref PubMed Scopus (332) Google Scholar, 21.Glick B.S. Cell. 1995; 80: 11-14Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 22.Pfanner N. Meijer M. Curr. Biol. 1995; 5: 132-135Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).On the other hand, mechanisms of protein translocation across the mitochondrial outer membrane are poorly understood. Translocation of many matrix-targeting precursor proteins, which do not fold in the intermembrane space, across the outer membrane can be likely driven by their passage across the inner membrane. However, at least for the initiation of the translocation process, precursor proteins have to move across the outer membrane by using only the outer membrane machinery until their presequences can interact with the inner membrane machinery. Then, what drives the initial translocation of the mitochondrial proteins across the outer membrane? It has been recently shown that isolated mitochondrial outer membrane vesicles without the inner membrane have the ability to translocate the presequence, but not the mature part, of precursor proteins(23.Mayer A. Lill R. Neupert W. J. Cell Biol. 1993; 121: 1233-1243Crossref PubMed Scopus (108) Google Scholar, 24.Mayer A. Neupert W. Lill R. Cell. 1995; 80: 127-137Abstract Full Text PDF PubMed Scopus (140) Google Scholar). The translocation of the presequence of the precursor proteins into the outer membrane vesicles involves at least two sites of presequence recognition at the outer membrane, one on the cis side and the other on the trans side (24.Mayer A. Neupert W. Lill R. Cell. 1995; 80: 127-137Abstract Full Text PDF PubMed Scopus (140) Google Scholar). Binding of the presequence to the “trans” site may promote unfolding of the precursor protein and translocation of the presequence across the outer membrane. An interesting hypothesis is that the acidic domain of MAS17 in the intermembrane space plays the role of the trans site; binding of the intermembrane space domain of MAS17 to the positively charged presequences of mitochondrial precursor proteins may pull precursors out of the import channel and into the mitochondria(15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar).In the present study, we examined the roles of the C-terminal acidic domain of MAS17 in protein import into mitochondria both in vivo and in vitro. The mutant MAS17 lacking the C-terminal acidic domain could mediate protein import into mitochondria as efficiently as wild-type MAS17. This suggests that the C-terminal domain of MAS17 is not essential for targeting and functions of MAS17.EXPERIMENTAL PROCEDURESStrains and PlasmidsThe following yeast strains and plasmids were used in this study. W303 (16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar) is a parental strain for constructing MNMS-1C, which has a disrupted chromosomal MAS17 gene but is rescued by pYE-Ura3:MAS17, where MAS17 is placed under GAL1 promoter. pYE-Ura3 (Clontech) and pRS314 (25.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) were used to introduce the wild-type and mutant MAS17 genes into yeast cells. MNMS-MAS17 and MNMS17Δ120-152 are MNMS-1C derivatives containing pRS314:MAS17 and pRS314:MAS17Δ120-152, respectively, instead of pYE-Ura3:MAS17. pUC119 (26.Sambrook J. Frithsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was used for site-directed mutagenesis of MAS17. Overproduction of MAS17 in Escherichia coli cells was performed using pET-21d (Novagen) as an expression plasmid and BL21(DE3) (Novagen) cells as host cells.Mutagenesis of the MAS17 GeneThe entire MAS17 coding region and its 5′-upstream and 3′-downstream regions were inserted into pUC119(26.Sambrook J. Frithsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Site-directed mutagenesis was then performed to construct mutant mas17 genes encoding MAS17Δ97-152 and MAS17Δ120-152; (i) AAC for Asn97 were changed to TAA for MAS17Δ97-152 and (ii) GAA for Glu120 were changed to TAG for MAS17Δ120-152. The mutant mas17 genes were inserted into pRS314, and the resulting pRS314:MAS17Δ97-152 and pRS314:MAS17Δ120-152 as well as the pRS314:MAS17 (for the wild-type MAS17 gene) were transformed into MNMS-1C cells. Fig. 1 shows the C-terminal regions of the truncated MAS17 proteins and wild-type MAS17.Production of AntibodiesMAS17 tagged with hexahistidine (MAS17-His6) was used as an antigen to immunize rabbits for raising antisera. For affinity purification of the anti-MAS17 antibodies, an antiserum was incubated with the polyvinylidene difluoride membrane that had preadsorbed MAS17 for 3 h, and the antibodies specific for MAS17 were eluted by the methods described previously(27.Johnson L.M. Snyder M. Chang L.M.S. Davis R.W. Campbell J.L. Cell. 1985; 43: 369-377Abstract Full Text PDF PubMed Scopus (100) Google Scholar).Isolation of Mitochondria and Import of Precursor Proteins in VitroMitochondria were prepared from yeast cells as described previously(28.Daum G. Böhni P.C. Schatz G. J. Biol. Chem. 1983; 257: 13028-13033Abstract Full Text PDF Google Scholar). In vitro transcription/translation was carried out for the following mitochondrial precursor proteins in the presence of [35S]methionine with rabbit reticulocyte lysate: the precursor of the F1-ATPase β subunit (pF1β), the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (pCOXIV-DHFR), and the presequence of the F0-ATPase subunit 9 fused to mouse dihydrofolate reductase (pSu9-DHFR). Denaturation of pCOXIV-DHFR was carried out by ammonium sulfate precipitation of the in vitro translation product followed by solubilization of the precipitates with 8 M urea and 10 mM Tris-HCl, pH 7.5. Each import reaction contained isolated yeast mitochondria (200 μg of protein) and 5 μl of rabbit reticulocyte lysate containing radioactively labeled precursor proteins in a total of 200 μl of import buffer (0.6 M mannitol, 50 mM HEPES-KOH, pH 7.4, 25 mM potassium Pi, pH 7.4, 120 mM KCl, 200 μg/ml methionine, 8 μg/ml pyruvate kinase (Sigma P1560), 1 mM dithiothreitol, 0.5 mM ATP, 0.5 mM MgCl2, and 5 mM phosphoenol pyruvate). After import was stopped by addition of valinomycin to 10 μg/ml, the reaction mixture was layered onto 500 μl of sucrose cushion (25% sucrose, 20 mM HEPES-KOH, pH 7.4, 2 mM EDTA), and the mitochondria were recovered by centrifugation for 10 min at 15,000 × g. Proteins in the mitochondrial pellets were analyzed by SDS-PAGE and fluorography, and were quantitated by laser densitometry.Trypsin Treatment of MitochondriaMitoplasts were prepared by incubating mitochondria in 20 mM HEPES-KOH, pH 7.4, for 30 min on ice and subsequent centrifugation. Trypsin treatment of mitochondria and mitoplasts was carried out as follows. Aliquots of mitochondria and mitoplasts (100 μg of protein) were suspended in 200 μl of 0.6 M mannitol, 20 mM HEPES-KOH, pH 7.4, and in 200 μl of 20 mM HEPES-KOH, pH 7.4, respectively. The suspension was incubated with various concentrations of trypsin (0, 40, 80, or 160 μg/ml) for 30 min on ice, and the digestion was terminated by adding 1 μl of 1 M phenylmethylsulfonyl fluoride. The mitochondria and mitoplasts were recovered by centrifugation, resuspended in 200 μl of 20 mM HEPES-KOH, pH 7.4, and subjected to trichloroacetic acid precipitation. Precipitated proteins were analyzed by SDS-PAGE and immunoblotting.Urea and Detergent Treatments of MitochondriaMitochondria (1 mg of protein) were suspended in 100 μl of 20 mM HEPES-KOH, pH 7.4, with 4 M urea for urea treatment or in 100 μl of 20 mM HEPES-KOH, pH 7.4, 250 mM NaCl, and 10% (w/v) glycerol with 0, 0.1, 0.25, 0.5, or 1.0% (w/v) digitonin for digitonin solubilization. After incubation for 30 min on ice, mitochondrial membranes and solubilized proteins were separated by centrifugation for 10 min at 200,000 × g.Miscellaneous MethodsStandard methods were used for yeast genetics(29.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar), DNA manipulation(26.Sambrook J. Frithsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), SDS-PAGE, and immunoblotting (30.Haid A. Suissa M. Methods Enzymol. 1983; 93: 192-205Crossref Scopus (231) Google Scholar). Fluorograms and immunoblots were quantified with a PD110 laser densitometer (Molecular Dynamics).RESULTSExpression of Truncated MAS17 in MAS17-deficient Yeast CellsMAS17 consists of three distinct domains: the N-terminal cytosolic domain (residues 1-94), the internal membrane-spanning domain (residues 95-119), and the C-terminal intermembrane space domain (residues 120-152). In order to analyze the functional role of each domain, we designed and constructed two mutant MAS17 genes, mas17Δ120-152 and mas17Δ97-152 encoding MAS17 lacking the intermembrane space domain (MAS17Δ120-152) and that lacking both the membrane spanning and intermembrane space domains (MAS17Δ97-152), respectively (Fig. 1).Since MAS17 is an essential gene of yeast(15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar, 16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar, 17.Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 2289-3382Crossref Scopus (112) Google Scholar), we first constructed a strain that expresses conditionally the MAS17 gene from the galactose-inducible GAL1 promoter. This strain, MNMS-1C, harbors a chromosomal disruption of the MAS17 gene, but is rescued by a single copy of MAS17 on a CEN4-ARS1-based plasmid (pYE-Ura3:MAS17). The pYE-Ura3:MAS17 plasmid contains the coding region of MAS17 fused to the yeast GAL1 promoter. The MNMS-1C strain could grow on galactose containing medium but not on medium supplemented with glucose (not shown). The two mutant mas17 genes, mas17Δ120-152 and mas17Δ97-152, as well as wild-type MAS17 and the vector alone, were separately transformed into the MNMS-1C cells carrying pYE-Ura3:MAS17, and the transformants were first selected on galactose-containing medium, where wild-type MAS17 was expressed from pYE-Ura3:MAS17. As shown in Fig. 2A, all the transformants could grow normally on galactose-containing medium. The transformants were then streaked onto glucose-containing medium where the expression of wild-type MAS17 was repressed, and abilities of the co-transformed mutant mas17 genes to complement the depletion of wild-type MAS17 were analyzed. Although cells bearing the mas17Δ97-152 gene could not grow on glucose-containing medium (Fig. 2B, sector 3), those carrying the mas17Δ120-152 gene could grow on glucose-containing medium as fast as those carrying the wild-type MAS17 gene (Fig. 2B, sectors 2 and 4). These results indicate that the membrane-spanning domain, but not the intermembrane space domain is essential for the function of MAS17 in vivo.Fig. 2Growth of yeast transformants harboring the wild-type or mutant MAS17 gene. A and B, the MNMS-1C cells carrying vector pRS314 alone (sector 1), pRS314:MAS17 (sector 2), pRS314:MAS17Δ97-152 (sector 3), or pRS314:MAS17Δ120-152 (sector 4) were streaked onto 2% galactose-containing medium (A), or 2% glucose-containing medium (B), and were incubated for 3 days at 30°C. C, the MNMS-MAS17 and the MNMS-MAS17Δ120-152 cells (see text) were inoculated in 2% glucose-containing or 3% glycerol containing medium, and cell growth in each culture was followed by monitoring the absorbance at 600 nm. All media contained 1% yeast extract and 2% polypeptone.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To analyze the ability of mas17Δ120-152 to complement the MAS17 deficiency more precisely, we isolated yeast cells that retained either the wild-type MAS17 gene (MNMS-MAS17) or the mas17Δ120-152 gene (MNMS-MAS17Δ120-152) on a pRS314 plasmid but had lost the pYE-Ura3:MAS17 plasmid. We then compared growth rates of the strains, MNMS-MAS17 and MNMS-MAS17Δ120-152 (Fig. 2C). The cells expressing MAS17Δ120-152 could grow nearly at the same rate as those with wild-type MAS17 under fermentable conditions (Fig. 2C (glucose)) and at a slightly lower rate than those with wild-type MAS17 under nonfermentable conditions (Fig. 2C (glycerol)). This means that MAS17Δ120-152 is almost fully functional in vivo.Subcellular and Submitochondrial Localization of the MAS17Δ120-152 ProteinWe next analyzed the assembly of the mutant MAS17 proteins in cells. An analysis of subcellular localization of MAS17 and MAS17Δ120-152 by immunoblotting with the anti-MAS17 antibodies showed both wild-type MAS17 and MAS17Δ120-152 were localized exclusively in the mitochondrial fraction but not in the post-mitochondrial supernatant (data not shown). We could not detect MAS17Δ97-152 by immunoblotting, because the anti-MAS17 antibodies we used recognize the membrane-spanning and intermembrane space domains, but not the cytosolic domain of MAS17 (not shown).In order to compare the topology of MAS17Δ120-152 in mitochondria with that of wild-type MAS17, we analyzed the sensitivity of MAS17Δ120-152 and MAS17 in intact mitochondria to trypsin (Fig. 3). As shown in Fig. 3A (a), treatment with 40 μg/ml of trypsin led to partial degradation of wild-type MAS17 to a fragment with an apparent size of 12 kDa (lane 2, asterisk). The fragment was hardly degraded even by 160 μg/ml of trypsin (lane 4) as long as intactness of the mitochondrial outer membrane was retained (Fig. 3A (b)). However, the 12-kDa fragment was degraded at lower concentrations of trypsin upon exposure of the intermembrane space by hypotonic swelling (Fig. 3A (a), lanes 6-8), excluding the possibility that it is intrinsically protease resistant. This 12-kDa fragment likely contains the C-terminal intermembrane space domain as shown for N. crassa MOM22 previously(14.Kiebler M. Keil P. Schneider H. van der Klei I.J. Pfanner N. Neupert W. Cell. 1993; 74: 483-492Abstract Full Text PDF PubMed Scopus (154) Google Scholar). Treatment with 40 μg/ml of trypsin also partially degraded MAS17Δ120-152 in intact mitochondria (Fig. 3B (a), lanes 1-4), suggesting that, like MAS17, MAS17Δ120-152 without the C-terminal domain is integrated into the outer membrane with its N-terminal domain exposed to the cytosol. As expected, in this case, trypsin treatment did not lead to the formation of the 12-kDa fragment (Fig. 3B (a), lanes 1-4).Fig. 3MAS17Δ120-152 and wild-type MAS17 are accessible to trypsin in intact mitochondria. Mitochondria and mitoplasts were prepared from the MNMS-MAS17 (A) and MNMS-MAS17Δ120-152 (B) strains and were treated with indicated concentrations of trypsin for 30 min on ice. After the proteolysis was stopped by addition of phenylmethylsulfonyl fluoride, mitochondria (lanes 1-4) and mitoplasts (lanes 5-8) were recovered by centrifugation. Proteins in each fraction were analyzed by SDS-PAGE and immunoblotting with anti-MAS17 (a), anti-Ssc1p and anti-cytochrome b2 antibodies (b). Protection of cytochrome b2 and Ssc1p against trypsin digestion reflect intactness of the mitochondrial outer membrane and of the outer and inner membranes, respectively. The asterisk indicates the 12-kDa fragment (see text).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Association of the MAS17Δ120-152 Protein with the Mitochondrial Outer MembranePeripherally bound membrane proteins can be extracted from membranes with salt (1 M NaCl), denaturant (4 M urea), or alkaline pH (0.2 M Na2CO3) while integral membrane proteins are not extracted with these treatments(31.Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar). We thus examined the nature of association of wild-type MAS17 and MAS17Δ120-152 with the mitochondrial outer membrane by treatment of mitochondria with 4 M urea. As shown in Fig. 4A, both MAS17Δ120-152 and MAS17 remained associated with the mitochondria upon extraction of peripheral membrane proteins with 4 M urea, suggesting that both proteins have characteristics of integral membrane proteins.Fig. 4Solubilization of MAS17Δ120-152 and wild-type MAS17 with urea and with digitonin from mitochondria. A, mitochondria (1 mg of protein) containing wild-type MAS17 or MAS17Δ120-152 were suspended in 100 μl of 20 mM HEPES-KOH, pH 7.4, with or without 4 M urea. After incubation for 30 min on ice, mitochondrial membranes were recovered by centrifugation at 20,0000 × g for 10 min. Proteins in the resulting pellet (P) and the supernatant (S) were analyzed by SDS-PAGE and immunoblotting with the anti-MAS17 antiserum. The total amounts recovered in both fractions (P + S) were taken as 100%. B, mitochondria containing wild-type MAS17 (a) or MAS17Δ120-152 (b) were treated with various concentrations of digitonin as described under “Experimental Procedures.” Proteins solubilized with indicated concentrations of digitonin were analyzed by SDS-PAGE and immunoblotting with the antibodies against MAS17, MAS20, ISP42, and MAS70. The amount of each protein in untreated mitochondria was taken as 100%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next solubilized mitochondrial membrane proteins with increasing concentrations of a detergent, digitonin (Fig. 4B). MAS17Δ120-152 was extracted with nearly the same concentration of digitonin as wild-type MAS17, suggesting that MAS17Δ120-152 is anchored to the mitochondrial outer membrane as tightly as wild-type MAS17. Interestingly, concentration dependencies of the solubilization of MAS17 and MAS17Δ120-152 parallel with ISP42 and MAS20, whereas MAS70 was extracted at lower concentrations of digitonin than MAS17, MAS17Δ120-152, ISP42, and MAS20. This is consistent with the model that MAS17 forms a core complex of the protein translocation machinery in the outer membrane with ISP42 and MAS20 but not with MAS70. 2M. Nakai, K. Kinoshita, and T. Endo, unpublished results. In Vitro Import of Precursor Proteins into Mitochondria Containing MAS17Δ120-152We then asked if replacement of MAS17 by MAS17Δ120-152 affects protein import into isolated mitochondria in vitro. We prepared wild-type mitochondria and MAS17Δ120-152-containing mitochondria as described above and tested in vitro import of several mitochondrial precursor proteins (Fig. 5). The pF1β was imported into the MAS17Δ120-152-containing mitochondria at a slightly slower rate than into the wild-type mitochondria. The rates of in vitro import of artificial mitochondrial precursor proteins, pCOXIV-DHFR and pSu9-DHFR, do not differ significantly between the wild-type and the MAS17Δ120-152-containing mitochondria (Fig. 5, B and C). If precursor unfolding or dissociation of the precursor from cytosolic factors represents a rate-limiting step of the protein import reaction under present assay conditions, a change in the rate of MAS17-mediating process, if any, could be masked. However, this is probably not the case, since urea-denatured pF1β (data not shown) and urea-denatured pCOXIV-DHFR (Fig. 5D) were imported into the MAS17Δ120-152-containing mitochondria almost as efficiently as into the wild-type mitochondria. These results suggest that the intermembrane space domain of MAS17 is not essential for protein translocation across the mitochondrial outer membrane in vitro.Fig. 5MAS17Δ120-152-containing mitochondria import precursor proteins as efficiently as wild-type mitochondria in vitro. Kinetics of in vitro import of precursor proteins were compared between mitochondria prepared from the MNMS-MAS17 strain (open circles) and those from the MNMS-MAS17Δ120-152 strain (closed circles) as described under “Experimental Procedures.” Import of pF1β (A) and pCOXIV-DHFR (B) were performed at 30°C, whereas import of pSu9-DHFR (C) and urea-denatured pCOXIV-DHFR (D) were at 16°C. Quantitative analyses were carried out after separation of mitochondrial proteins by SDS-PAGE and fluorography, followed by densitometry scanning of the mature forms.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn the present study, we examined the role of the acidic C-terminal domain of MAS17 in protein translocation across the mitochondrial outer membrane both in vivo and in vitro. The strain containing mutant MAS17 (MAS17Δ120-152) that lacks the intermembrane space domain grew as fast as the wild-type strain under fermentable or nonfermentable conditions. MAS17Δ120-152 was integrated into the mitochondrial outer membrane with the same topology as wild-type MAS17. Mitochondria containing MAS17Δ120-152 could import several mitochondrial precursor proteins as efficiently as those containing wild-type MAS17 in vitro. A possible defect of MAS17Δ120-152 may have been perhaps suppressed by increase in the amount(s) of other component(s) of mitochondrial protein translocation machineries. However, this is probably not the case, since replacement of MAS17 by MAS17Δ120-152 in mitochondria did not significantly affect the amounts of such proteins as MAS20 and MAS70, receptor proteins in the outer membrane, ISP42, the component of the translocation machinery in the outer membrane, MIM17, MIM23, and MIM44, the components of the translocation machinery in the inner membrane (32.Pfanner N. Craig E.A. Meijer M. Trends Biochem. Sci. 1994; 19: 368-372Abstract Full Text PDF PubMed Scopus (112) Google Scholar), and Ssc1p, mitochondrial hsp70 in the matrix that mediates protein translocation across the inner membrane (not shown). Taken together, we conclude that the C-terminal domain of MAS17 is not essential for its role in protein import into mitochondria. This means that, although MAS17 may facilitate protein translocation across the outer membrane by binding to precursor proteins on the cytosolic side of the membrane, it does not pull the precursor protein from the trans side of the outer membrane.Targeting signals of mitochondrial outer membrane proteins that have a single transmembrane segment near the N terminus or the C terminus were studied previously. In the case of MAS70 with a transmembrane segment near the N terminus, the positively charged, extreme N-terminal region functions as a targeting sequence, whereas the subsequent transmembrane segment is necessary for stop-transfer and anchoring functions(33.Nakai M. Hase T. Matsubara H. J. Biochem. (Tokyo). 1989; 105: 513-519Crossref PubMed Scopus (24) Google Scholar). The transmembrane segment of MAS70 alone is also capable of targeting and inserting a passenger protein(34.McBride H.M. Millar D.G. Li J.-M Shore G.C. J. Cell Biol. 1992; 119: 1451-1457Crossref PubMed Scopus (85) Google Scholar). In the case of the Bcl-2 protein with a transmembrane segment near the C terminus, the C-terminal region, including the transmembrane segment function as a signal-anchor sequence selective for the mitochondrial outer membrane, whereas association with the endoplasmic reticulum and nuclear envelope occurs by a different mechanism(35.Nakai M. Takeda A. Clearly M.L. Endo T. Biochem. Biophys. Res. Commun. 1993; 196: 233-239Crossref PubMed Scopus (54) Google Scholar, 36.Nguyen M. Millar D.G. Yong V.W. Korsmeyer S.J. Shore G.C. J. Biol. Chem. 1993; 268: 25265-25268Abstract Full Text PDF PubMed Google Scholar). MAS17 is unique in that it has a transmembrane segment in the middle of the polypeptide chain. The present study shows that correct targeting of MAS17 to the mitochondria was not impaired by deletion of the C-terminal intermembrane space domain, suggesting that it does not encompass the targeting signal of MAS17 for the mitochondrial outer membrane. Since in vitro import of N. crassa MOM22 into isolated mitochondria strictly depends on the surface receptor, MOM72 and MOM19(37.Keil P. Pfanner N. FEBS Lett. 1993; 321: 197-200Crossref PubMed Scopus (62) Google Scholar), identification of the targeting signal of MAS17 should be important for understanding the substrate specificity in the recognition of mitochondrial outer membrane proteins by the mitochondrial protein receptors. INTRODUCTIONNuclear-encoded mitochondrial precursor proteins are imported from the cytosol into mitochondria; they are recognized by cytosolic factors and/or mitochondrial receptor proteins and are subsequently translocated across the outer and inner mitochondrial membranes(1.Segui-Real B. Stuart R.A. Neupert W. FEBS Lett. 1992; 313: 2-7Crossref PubMed Scopus (44) Google Scholar, 2.Schatz G. Protein Sci. 1993; 2: 141-146Crossref PubMed Scopus (72) Google Scholar). Translocation across the mitochondrial membranes requires coordinated functions of two separate machineries in the outer and inner mitochondrial membranes; they most likely interact transiently to allow the movement of precursor proteins into the matrix(3.Horst M. Hilfiker-Rothenfluh S. Oppliger W. Schatz G. EMBO J. 1995; 14: 2293-2297Crossref PubMed Scopus (83) Google Scholar, 4.Berthold J. Bauer M.F. Schneider H.-C. Klaus C. Dietmeier K. Neupert W. Brunner M. Cell. 1995; 81: 1085-1093Abstract Full Text PDF PubMed Scopus (154) Google Scholar).MAS20 (5.Hines V. Brandt A. Griffiths G. Horstmann H. Brütsch H. Schatz G. EMBO J. 1990; 9: 3191-3200Crossref PubMed Scopus (203) Google Scholar) and MAS70 (6.Ramage L. Junne T. Hahne K. Lithgow T. Schatz G. EMBO J. 1993; 12: 4115-4123Crossref PubMed Scopus (180) Google Scholar) in Saccharomyces cerevisiae and MOM19 (7.Söllner T. Griffiths G. Pfaller R. Pfanner N. Neupert W. Cell. 1989; 59: 1061-1070Abstract Full Text PDF PubMed Scopus (247) Google Scholar) and MOM72 (8.Söllner T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 1990; 62: 107-115Abstract Full Text PDF PubMed Scopus (216) Google Scholar) in Neurospora crassa serve as receptor proteins and are responsible for the initial binding of mitochondrial precursor proteins to the mitochondrial surface. Yeast MAS37 may form a heterodimer complex with MAS70 and facilitate the receptor function of MAS70(9.Gratzer S. Lithgow T. Bauer R.E. Lamping E. Paltauf F. Kohlwein S.D. Haucke V. Junne T. Schatz G. Horst M. J. Cell Biol. 1995; 129: 25-34Crossref PubMed Scopus (152) Google Scholar). ISP42 in yeast (10.Baker K. Schaniel A. Vestweber D. Schatz G. Nature. 1990; 348: 605-609Crossref PubMed Scopus (195) Google Scholar) and MOM38 in N. crassa(11.Kiebler M. Pfaller R. Söllner T. Griffiths G. Horstmann H. Pfanner N. Neupert W. Nature. 1990; 348: 610-616Crossref PubMed Scopus (192) Google Scholar) likely mediate the step of subsequent protein translocation across the outer membrane. ISP42/MOM38 was found to be in contact with precursor proteins arrested in transit across the mitochondrial membranes(12.Vestweber D. Brunner J. Baker A. Schatz G. Nature. 1989; 341: 205-209Crossref PubMed Scopus (192) Google Scholar, 13.Söllner T. Rassow J. Wiedmann M. Schlossmann J. Keil P. Neupert W. Pfanner N. Nature. 1992; 355: 84-87Crossref PubMed Scopus (129) Google Scholar), and antibodies against ISP42 blocked protein import into mitochondria(12.Vestweber D. Brunner J. Baker A. Schatz G. Nature. 1989; 341: 205-209Crossref PubMed Scopus (192) Google Scholar).Another outer membrane protein MOM22 in N. crassa appears to function downstream of the receptors because antibodies against MOM22 inhibited import of mitochondrial precursor proteins into mitochondria but not their binding to receptors in vitro(14.Kiebler M. Keil P. Schneider H. van der Klei I.J. Pfanner N. Neupert W. Cell. 1993; 74: 483-492Abstract Full Text PDF PubMed Scopus (154) Google Scholar). MAS17 (MAS22), the yeast equivalent of N. crassa MOM22, has been identified recently(15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar, 16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar, 17.Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 2289-3382Crossref Scopus (112) Google Scholar). The MAS17 gene is essential (15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar, 16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar, 17.Hönlinger A. Kübrich M. Moczko M. Gärtner F. Mallet L. Bussereau F. Eckerskorn C. Lottspeich F. Dietmeier K. Jacquet M. Pfanner N. Mol. Cell. Biol. 1995; 15: 2289-3382Crossref Scopus (112) Google Scholar) and depletion of functional MAS17 results in accumulation of the precursor form of a mitochondrial protein(16.Nakai M. Endo T. FEBS Lett. 1995; 357: 202-206Crossref PubMed Scopus (43) Google Scholar). MAS17 consists of three distinct domains: the N-terminal domain that is highly acidic and faces the cytosol, the internal hydrophobic domain that is integrated into the outer membrane, and the C-terminal domain that contains several acidic residues and faces the intermembrane space. The cytosolic acidic domain may well provide a binding site for positively charged presequences of mitochondrial precursor proteins.A key question concerning the protein import into mitochondria is what drives the vectorial movement of precursor proteins across the two membranes. In the case of protein translocation across the inner mitochondrial membrane, the membrane potential across the inner membrane triggers the movement of a positively charged presequence across the inner membrane probably by electrophoretic effects(18.Roise D. Horvath S.J. Tomich J.M. Richards J.H. Schatz G. EMBO J. 1986; 5: 1327-1334Crossref PubMed Scopus (308) Google Scholar, 19.Martin J. Mahlke K. Pfanner N. J. Biol. Chem. 1991; 266: 18051-18057Abstract Full Text PDF PubMed Google Scholar). Then mitochondrial hsp70 1The abbreviations used are: hsp7070-kDa heat shock proteinpF1βthe precursor of the F1-ATPase β subunitpCOXIV-DHFRthe presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductasepSu9-DHFRthe presequence of the F0-ATPase subunit 9 fused to mouse dihydrofolate reductasePAGEpolyacrylamide gel electrophoresis. likely drives the movement of the rest of the precursor polypeptide chain at the expense of ATP hydrolysis in the matrix(20.Schneider H.-C. Berthold J. Bauer M.F. Dietmeier K. Guiard B. Bruner M. Neupert W. Nature. 1994; 371: 768-774Crossref PubMed Scopus (332) Google Scholar, 21.Glick B.S. Cell. 1995; 80: 11-14Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 22.Pfanner N. Meijer M. Curr. Biol. 1995; 5: 132-135Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).On the other hand, mechanisms of protein translocation across the mitochondrial outer membrane are poorly understood. Translocation of many matrix-targeting precursor proteins, which do not fold in the intermembrane space, across the outer membrane can be likely driven by their passage across the inner membrane. However, at least for the initiation of the translocation process, precursor proteins have to move across the outer membrane by using only the outer membrane machinery until their presequences can interact with the inner membrane machinery. Then, what drives the initial translocation of the mitochondrial proteins across the outer membrane? It has been recently shown that isolated mitochondrial outer membrane vesicles without the inner membrane have the ability to translocate the presequence, but not the mature part, of precursor proteins(23.Mayer A. Lill R. Neupert W. J. Cell Biol. 1993; 121: 1233-1243Crossref PubMed Scopus (108) Google Scholar, 24.Mayer A. Neupert W. Lill R. Cell. 1995; 80: 127-137Abstract Full Text PDF PubMed Scopus (140) Google Scholar). The translocation of the presequence of the precursor proteins into the outer membrane vesicles involves at least two sites of presequence recognition at the outer membrane, one on the cis side and the other on the trans side (24.Mayer A. Neupert W. Lill R. Cell. 1995; 80: 127-137Abstract Full Text PDF PubMed Scopus (140) Google Scholar). Binding of the presequence to the “trans” site may promote unfolding of the precursor protein and translocation of the presequence across the outer membrane. An interesting hypothesis is that the acidic domain of MAS17 in the intermembrane space plays the role of the trans site; binding of the intermembrane space domain of MAS17 to the positively charged presequences of mitochondrial precursor proteins may pull precursors out of the import channel and into the mitochondria(15.Lithgow T. Junne T. Suda K. Gratzer S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11973-11977Crossref PubMed Scopus (142) Google Scholar).In the present study, we examined the roles of the C-terminal acidic domain of MAS17 in protein import into mitochondria both in vivo and in vitro. The mutant MAS17 lacking the C-terminal acidic domain could mediate protein import into mitochondria as efficiently as wild-type MAS17. This suggests that the C-terminal domain of MAS17 is not essential for targeting and functions of MAS17." @default.
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