Characterization of Peptides Released from Mitochondria
2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês
10.1074/jbc.m410609200
ISSN1083-351X
AutoresSteffen Augustin, Mark Nolden, Stefan Müller, Olaf Hardt, Isabel Arnold, Thomas Langer,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoConserved ATP-dependent proteases ensure the quality control of mitochondrial proteins and control essential steps in mitochondrial biogenesis. Recent studies demonstrated that non-assembled mitochondrially encoded proteins are degraded to peptides and amino acids that are released from mitochondria. Here, we have characterized peptides extruded from mitochondria by mass spectrometry and identified 270 peptides that are exported in an ATP- and temperature-dependent manner. The peptides originate from 51 mitochondrially and nuclearly encoded proteins localized mainly in the matrix and inner membrane, indicating that peptides generated by the activity of all known mitochondrial ATP-dependent proteases can be released from the organelle. Pulse-labeling experiments in logarithmically growing yeast cells revealed that ∼6–12% of preexisting and newly imported proteins is degraded and contribute to this peptide pool. Under respiring conditions, we observed an increased proteolysis of newly imported proteins that suggests a higher turnover rate of respiratory chain components and thereby rationalizes the predominant appearance of representatives of this functional class in the detected peptide pool. These results demonstrated a constant efflux of peptides from mitochondria and provided new insight into the stability of the mitochondrial proteome and the efficiency of mitochondrial biogenesis. Conserved ATP-dependent proteases ensure the quality control of mitochondrial proteins and control essential steps in mitochondrial biogenesis. Recent studies demonstrated that non-assembled mitochondrially encoded proteins are degraded to peptides and amino acids that are released from mitochondria. Here, we have characterized peptides extruded from mitochondria by mass spectrometry and identified 270 peptides that are exported in an ATP- and temperature-dependent manner. The peptides originate from 51 mitochondrially and nuclearly encoded proteins localized mainly in the matrix and inner membrane, indicating that peptides generated by the activity of all known mitochondrial ATP-dependent proteases can be released from the organelle. Pulse-labeling experiments in logarithmically growing yeast cells revealed that ∼6–12% of preexisting and newly imported proteins is degraded and contribute to this peptide pool. Under respiring conditions, we observed an increased proteolysis of newly imported proteins that suggests a higher turnover rate of respiratory chain components and thereby rationalizes the predominant appearance of representatives of this functional class in the detected peptide pool. These results demonstrated a constant efflux of peptides from mitochondria and provided new insight into the stability of the mitochondrial proteome and the efficiency of mitochondrial biogenesis. Mitochondria are dynamic organelles whose number, shape, and protein composition varies in different metabolic and differentiation states (1Shaw J.M. Nunnari J. Trends Cell Biol. 2002; 12: 178-184Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Although a number of signaling pathways have been identified that allow the coordination of mitochondrial and nuclear gene expression systems under varying growth conditions (2Costanzo M.C. Fox T.D. Ann. Rev. Genet. 1990; 24: 91-113Crossref PubMed Google Scholar, 3Butow R.A. Avadhani N.G. Mol. Cell. 2004; 14: 1-15Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar, 4Zhao Q. Wang J. Levichkin I.V. Stasinopoulos S. Ryan M.T. Hoogenraad N.J. EMBO J. 2002; 21: 4411-4419Crossref PubMed Scopus (703) Google Scholar, 5Yoneda T. Benedetti C. Urano F. Clark S.G. Harding H.P. Ron D. J. Cell Sci. 2004; 117: 4055-4066Crossref PubMed Scopus (413) Google Scholar), next to nothing is known about the turnover of mitochondrial proteins and the stability of the mitochondrial proteome. Mitochondria harbor a conserved proteolytic system capable of degrading polypeptides to amino acids and therefore have been considered to be a final destination for proteins (6Desautels M. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1869-1873Crossref PubMed Scopus (87) Google Scholar). Various ATP-dependent proteases ensure the quality control of mitochondrial proteins in different subcompartments and regulate the biogenesis of the organelle (7Bota D.A. Davies K.J.A. Mitochondrion. 2001; 1: 33-49Crossref PubMed Scopus (90) Google Scholar, 8Van Dyck L. Langer T. Cell. Mol. Life Sci. 1999; 55: 825-842Crossref Scopus (86) Google Scholar). They are thought to degrade proteins to peptides that are subsequently degraded to amino acids by only poorly characterized oligopeptidases within mitochondria. The first evidence for a release of peptides from mitochondria came from the observation that peptides derived from mitochondrially encoded proteins were detected at the cell surface of mammalian cells in association with major histocompatibility antigen class I molecules (9Loveland B. Wang C.R. Yonekawa H. Hermel E. Lindahl K.F. Cell. 1990; 60: 971-980Abstract Full Text PDF PubMed Scopus (308) Google Scholar). The analysis of the proteolytic breakdown of non-assembled mitochondrial translation products in yeast indeed revealed the quantitative release of degradation products from the organelle (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). Whereas free amino acids represented ∼70% of the degradation products and are most likely exported from mitochondria by various amino acid transporters in the inner membrane (11Wipf D. Ludewig U. Tegeder M. Rentsch D. Koch W. Frommer W.B. Trends Biochem. Sci. 2002; 27: 139-147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), two release pathways were identified for peptides composed of 6–20 amino acid residues (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). The majority of mitochondrial translation products is degraded by the m-AAA protease, a membrane-bound ATP-dependent proteolytic complex exposing its catalytic sites to the matrix (12Arlt H. Tauer R. Feldmann H. Neupert W. Langer T. Cell. 1996; 85: 875-885Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Peptides generated by the yeast m-AAA protease are released to the matrix and transported across the inner membrane by the ABC transporter Mdl1 (13Dean M. Allikmets R. Gerrard B. Stewart C. Kistler A. Shafer B. Michaelis S. Strathern J. Yeast. 1994; 10: 377-383Crossref PubMed Scopus (58) Google Scholar), a homologue of the transporter associated with antigen presentation (TAP) in the endoplasmic reticulum (14Borst P. Elferink R.O. Ann. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1341) Google Scholar). The i-AAA protease, on the other hand, releases peptides generated upon proteolysis of mitochondrial translation products into the intermembrane space (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). This conserved ATP-dependent proteolytic complex resides in the inner membrane and is composed of Yme1 subunits that expose catalytic sites to the intermembrane space (15Weber E.R. Hanekamp T. Thorsness P.E. Mol. Biol. Cell. 1996; 7: 307-317Crossref PubMed Scopus (119) Google Scholar, 16Leonhard K. Herrmann J.M. Stuart R.A. Mannhaupt G. Neupert W. Langer T. EMBO J. 1996; 15: 4218-4229Crossref PubMed Scopus (218) Google Scholar). Although these experiments established that peptides are set free from mitochondria, the extent of peptide export as well as the physiological function of released peptides remained unclear. In view of the capability of the mitochondrial proteolytic system to completely degrade polypeptides to amino acid residues, it seems unlikely that mitochondrial peptides are transported to the cytosol for their destruction. Yeast cells lacking the ABC transporter Mdl1 do not exhibit growth deficiencies under non-stress conditions, in agreement with the observation that mitochondrial peptide export is only partially impaired in these cells (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). Overexpression of Mdl1, on the other hand, increases the sensitivity of yeast cells to reactive oxygen species linking the function of Mdl1 to the cellular resistance toward oxidative stress (17Chloupkova M. LeBard L.S. Koeller D.M. J. Mol. Biol. 2003; 331: 155-165Crossref PubMed Scopus (75) Google Scholar). Moreover, Mdl1 associates with the F1FO-ATP synthase in the inner membrane in a nucleotide-dependent manner (18Galluhn D. Langer T. J. Biol. Chem. 2004; 279: 38338-38845Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), raising the intriguing possibility that peptide export from mitochondria is coupled to the activity of the F1FO-ATP synthase and thereby to the cellular energy metabolism. As only the degradation of non-assembled mitochondrial translation products has been analyzed, it remained unclear whether peptide export is restricted to mitochondrially encoded substrates of AAA proteases in the inner membrane or also includes matrix-localized substrates of other peptidases. Moreover, the stability of the mitochondrial proteome and the extent of peptide export in yeast cells have not been studied so far. In this study, we have therefore analyzed the proteolysis of newly synthesized and preexisting mitochondrial proteins and characterized peptides exported from mitochondria by mass spectrometry. Yeast Strains and Growth Conditions—Cells were grown at 30 °C in YP medium containing glucose (2%) or galactose (2%) or on lactate medium. All strains were derivatives of W303–1A. The Δyme1 strain has been described (16Leonhard K. Herrmann J.M. Stuart R.A. Mannhaupt G. Neupert W. Langer T. EMBO J. 1996; 15: 4218-4229Crossref PubMed Scopus (218) Google Scholar). For generation of the strains Δmdl1 (YIA29) and Δmdl1Δyme1 (YIA31), MDL1 and YME1 were deleted by PCR-mediated homologous recombination using a HIS3MX6 and a KanMX6 cassette, respectively. Similarly, the genomic NDE1 gene was modified using a 3HA-HIS3MX6 cassette to allow the expression of a carboxyl-terminal HA-tagged variant of Nde1 in wild type (YSA1) or Δyme1 (YSA2) cells. Monitoring Peptide Export from Mitochondria—Mitochondria were isolated by differential centrifugation, further purified using a sucrose density gradient (19Meisinger C. Sommer T. Pfanner N. Anal. Biochem. 2000; 287: 339-342Crossref PubMed Scopus (123) Google Scholar), and resuspended in buffer A (0.6 M sorbitol, 150 mm KCl (pH 7.4), 15 mm potassium phosphate buffer (pH 7.4), 20 mm Tris/HCl (pH 7.4), 13 mm MgSO4, 4 mm ATP, 0.5 mm GTP, 3 mm amino acid mix (including all proteinogenic amino acids except methionine, cysteine, tyrosine), 66 μm cysteine, 7 μm tyrosine, 6 mm α-ketoglutarate, 5 mm phosphoenolpyruvate, pyruvate kinase (0.04 mg/mg mitochondria)). After two additional washing steps using 1 ml of SHKCl buffer (0.6 mm sorbitol, 50 mm HEPES/KOH, pH 7.2, 80 mm KCl), mitochondria (13.5 mg) were incubated in buffer A (lacking pyruvate kinase) at a concentration of 27 mg/ml for 30 min at 37 or 30 °C to allow proteolysis to occur. Samples were split into supernatant and pellet fractions by centrifugation at 4 °C for 4 min at 16,100 × g. The integrity of the organelles was assessed by immunoblotting using various mitochondrial marker proteins. For ATP depletion, mitochondria were incubated prior to washing for 5 min at 25 °C in buffer A containing apyrase (0.12 units/mg mitochondria) and oligomycin (20 μg/mg mitochondria), but lacking ATP, GTP, α-ketoglutarate, and the ATP regenerating system. After repeated washing, mitochondria were further incubated in the presence of apyrase (0.04 units/mg mitochondria) and oligomycin (2 μg/mg mitochondria). Mitochondrial supernatants (500 μl) were subjected to sizing chromatography at 12 °C using a Sephadex peptide column (7.5/300; Amersham Biosciences) as described (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). For calibration, mitochondrial translation products were labeled with [35S]methionine in isolated mitochondria in parallel experiments. Eluate fractions containing peptides composed of 6–20 amino acid residues were identified. The corresponding fractions of the large scale preparations were collected and concentrated in an evaporation centrifuge. Peptides in the tube were resuspended in 20 μl of 0.1% (v/v) trifluoroacetic acid and further analyzed by liquid chromatography-MS/MS. 1The abbreviations used are: MS, mass spectrometry; HA, hemagglutinin; IM, mitochondrial inner membrane; OM, mitochondrial outer membrane. Control experiments suggested that ∼45% of the detected larger peptides was recovered under these conditions (data not shown). Mass Spectrometry—Liquid chromatography-MS/MS data were acquired on a Q-Tof II quadrupole-time of flight mass spectrometer (Micromass). Samples were introduced using the Ultimate nano-LC system (LC Packings). The column setup comprised a 0.3 mm × 1-mm trap column and a 0.075 × 150-mm analytical column, both packed with 3 μm PepMap C18 (LC Packings). Samples (8 μl) were desalted on a trap column for 1 min using 0.1% trifluoroacetic acid at a flow rate of 30 μl/min. Peptides were eluted onto the analytical column using a gradient of 5–40% acetonitrile in 0.1% trifluoroacetic acid over 90 min at a column flow rate of ∼200 nl/min. The analytical column and the spray emitter (Carbotech) were connected directly, and stable nanospray was established by the application of 1.7–2.0 kV to the distal end of the column. Data-dependent acquisition of MS and MS/MS spectra was controlled by Masslynx software (Micromass). Survey scans of 1 s covered the range from m/z 400 to m/z 1400. Doubly charged ions were selected for MS/MS experiments. In MS/MS mode the mass range from m/z 40 to m/z 1400 was scanned. Micromass-formatted peak lists were generated from the raw data using the Proteinlynx software module; proteins were identified by fragment ion searches against the NCBI non-redundant data base using version 1.9 of the Mascot search engine. Data Evaluation—All assignments with a probability score >30 and average mass deviations of fragment ions 30. Proteins were only included in the data set if corresponding peptides were identified in at least two of four experiments. A protein was classified as mitochondrial or non-mitochondrial according to published data. The submitochondrial localization is given as previously demonstrated or was predicted based on the following criteria: Only proteins containing at least one predicted transmembrane domain were classified as OM or IM proteins. All peripheral membrane proteins were localized to the intermembrane space or matrix. Functional classification of mitochondrial proteins was based on the functional categories used by the MitoP2 data base (20Andreoli C. Prokisch H. Hortnagel K. Mueller J.C. Munsterkotter M. Scharfe C. Meitinger T. Nucleic Acids Res. 2004; 32: D459-D462Crossref PubMed Google Scholar). Mitochondrial transcriptome data were retrieved from Ref. 21Ohlmeier S. Kastaniotis A.J. Hiltunen J.K. Bergmann U. J. Biol. Chem. 2004; 279: 3956-3979Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar. Analysis of the Stability of Mitochondrial Proteins—Yeast cells (8 A600 units) were grown at 30 °C in minimal medium supplemented with the carbon sources indicated and labeled with [35S]methionine (300 μCi) either for 14 h or for 10 min. When indicated, mitochondrial or cytosolic protein synthesis was inhibited by incubating cells prior to labeling with chloramphenicol (6 mg/ml) or cycloheximide (1 mg/ml), respectively, for 15 min at 30 °C. Incorporation of [35S]methionine into polypeptides was assessed by the accumulation of trichloroacetic acid-insoluble radioactivity. Mitochondria were isolated as described above, resuspended in buffer A, and then washed four times with 0.5 ml of cold SHKCl buffer. The mitochondrial pellet was resuspended carefully in buffer A (lacking pyruvate kinase) and split into four aliquots that were incubated at 37 °C. At the time points indicated, samples were centrifuged immediately at 4 °C for 4 min at 16,100 × g. The radioactivity present in pellet and supernatant fractions was determined. Stability of Nde1 and Cox2 within Mitochondria—After addition of cycloheximide (1 mg/ml) or chloramphenicol (6 mg/ml) to yeast cells growing logarithmically in lactate medium at 30 °C, cultures were shifted to 37 °C. At the time points indicated, aliquots (10 A600 units) were withdrawn and cells disrupted by glass beads. Mitochondrial membranes were isolated by differential centrifugation and analyzed by SDS-PAGE and immunoblotting using antisera directed against Cox2 and Nde1. The peptide C-KNLMTKLEEQDSRRG of Nde1 was used for generation of antibodies in rabbits. Proteolytic Products Derived from Both Mitochondrially and Nuclearly Encoded Polypeptides Are Released from Mitochondria—In an attempt to further characterize the peptides exported from mitochondria, we first tested whether degradation products of nuclearly encoded mitochondrial proteins are extruded from the organelle. Yeast cells logarithmically growing on galactose-containing medium were labeled for 10 min with [35S]methionine. Mitochondria were isolated and incubated at various temperatures to examine the release of radioactivity from the organelle (Fig. 1A). We observed a time- and temperature-dependent accumulation of radioactivity in the supernatant (Fig. 1A). At 24 °C, ∼3% of the incorporated [35S]methionine was set free from mitochondria, whereas this value increased to ∼6% at 37 °C (Fig. 1A). A similar amount of radioactivity was released from mitochondria isolated from cells grown on glucose-containing medium (data not shown; see also Fig. 6A). In agreement with our previous findings (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar), these data indicate proteolysis of mitochondrial proteins. Incorporated [35S]methionine was quantitatively recovered from the trichloroacetic acid-insoluble fraction of a trichloroacetic acid precipitation, whereas the radioactive material released from mitochondria is trichloroacetic acid-soluble, i.e. represents small peptides and amino acid residues (data not shown).Fig. 6Stability of mitochondrial proteins under respiring and fermenting conditions. Yeast cells were grown at 30 °C on glucose-(▴) or on lactate-(▪) containing media. A, to monitor the turnover of the mitochondrial proteome, cells were labeled with [35S]methionine overnight. B, to determine the stability of proteins newly imported into mitochondria, cells were radiolabeled for 10 min only. The release of degradation products from isolated mitochondria was assessed at 37 °C as in Fig. 1. Radioactivity in the mitochondrial supernatant fraction is given as percentage of total incorporated radioactivity in both pellet and supernatant fraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT) If the radioactivity released from mitochondria were exclusively generated by the proteolytic breakdown of mitochondrial translation products, radioactive material should not accumulate in the supernatant fraction upon inhibition of mitochondrial protein synthesis by chloramphenicol. However, the release of radioactivity from mitochondria was not significantly affected by the presence of chloramphenicol (Fig. 1B). Control experiments revealed that mitochondrial protein synthesis was inhibited under these conditions (Fig. 1C). We have concluded that degradation products of mitochondrially encoded polypeptides represent only a minor fraction of the material released from the organelle. Rather, nuclearly encoded mitochondrial proteins apparently are another source of the released radioactivity. Stability of the Mitochondrial Proteome Assessed by Two-dimensional Gel Electrophoresis—These experiments indicated that, in logarithmically growing yeast cells at 37 °C, ∼6% of newly synthesized nuclearly as well as mitochondrially encoded proteins is degraded, followed by the release of the proteolytic breakdown products from the organelle. Proteolysis may be caused by the limited efficiency of mitochondrial biogenesis in general, including import and folding reactions of a large number of proteins. Alternatively, the degradation of a specific subset of mitochondrial proteins may be responsible for the release of a portion of the incorporated [35S]methionine from the organelle. To distinguish between these possibilities, we analyzed the stability of the mitochondrial proteome by two-dimensional gel electrophoresis. Highly purified mitochondria were obtained from logarithmically growing yeast cells and analyzed prior to or after a further incubation for 30 min at 37 °C to allow proteolysis to occur. In parallel, mitochondria were purified after pulse-labeling of yeast cells with [35S]methionine to examine the stability of newly synthesized mitochondrial proteins. In agreement with previous findings (22Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schonfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13207-13212Crossref PubMed Scopus (712) Google Scholar), ∼280 protein spots were identified on two-dimensional gels. For quantification of individual protein spots, we compared master gels generated after each electrophoresis had been repeated at least three times. These experiments revealed a remarkable stability of the mitochondrial proteome and did not allow the identification of individual protein spots that were significantly decreased or disappeared after an incubation of the mitochondria at 37 °C (data not shown; see supplemental information). It therefore appears likely that the degradation of a small fraction of a large number, rather than a significant degradation of a small number of polypeptides, accounts for the observed proteolysis of 35S-labeled mitochondrial proteins. Defining the Origin of Peptides Released from Mitochondria— The unambiguous identification of the source of exported proteolytic breakdown products requires the characterization of the peptides released from mitochondria. We therefore performed peptide export experiments on a large scale. Mitochondria were isolated from yeast cells logarithmically growing on non-fermentable carbon sources, and peptides accumulating in the supernatant were fractionated by a peptide gel filtration column. Fractions with peptides consisting of >6 amino acid residues were pooled and analyzed by liquid chromatography-MS/MS. To take statistical variations into account, each experiment was repeated four times, and only peptides identified in at least two of four fractionations were considered. These experiments led to the identification of 270 different peptides released from mitochondria that correspond to 51 different proteins (Table I and see supplemental information). 82% of these proteins has been previously localized to mitochondria by various methods, whereas the subcellular localization of 6% has not been determined (Fig. 2A and Table I). Six of the identified peptide sources correspond to non-mitochondrial proteins. Although three of them have also been detected in mitochondria by large scale tandem mass spectrometry (22Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schonfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13207-13212Crossref PubMed Scopus (712) Google Scholar), they most likely represent contaminations of our preparation.Table IProtein sources of released peptidesProteinNo. of peptidesSequence coverageNo. of runsGenomeLocalizationClassificationCellularMit.Nde13454.34 of 4NucmIMRespiratory chainCox22855.44 of 4MitmIMRespiratory chainYlr356w1438.14 of 4NucmNDNDGut2613.44 of 4NucmIMCarbohydrate metabolismCox35194 of 4MitmIMRespiratory chainStf1439.54 of 4NucmMRespiratory chainAco146.74 of 4NucmMCarbohydrate metabolismAtp18340.72 of 4NucmIMRespiratory chainAtp17339.62 of 4NucmIMRespiratory chainAtp19325.83 of 4NucmIMRespiratory chainSdh4322.72 of 4NucmIMRespiratory chainOm45322.14 of 4NucmOMNDIdh1313.64 of 4NucmMCarbohydrate metabolismAtp737.54 of 4NucmMRespiratory chainKgd235.42 of 4NucmMCarbohydrate metabolismYmr31225.23 of 4NucmMProtein translation/stabilityGrx5216.74 of 4NucmMDetox./protect. enzymesAtp4212.34 of 4NucmIMRespiratory chainMpm129.52 of 4NucmNDNDRsm2827.53 of 4NucmMProtein translation/stabilityCox126.74 of 4MitmIMRespiratory chainCob24.72 of 4MitmIMRespiratory chainIlv223.92 of 4NucmMaa and nitrogen metabolismTim11116.73 of 4NucmIMRespiratory chainAtp14116.12 of 4NucmMRespiratory chainImg2114.44 of 4NucmMProtein translation/stabilityCox12113.34 of 4NucmIM (p)Respiratory chainYbr230c112.72 of 4NucmNDNDQcr71112 of 4NucmMRespiratory chainRim1110.42 of 4NucmMNucleic acid metabolismTim12110.13 of 4NucmIMSProtein sortingSdh317.14 of 4NucmIMRespiratory chainRip116.53 of 4NucmIMRespiratory chainAtp314.82 of 4NucmMRespiratory chainCor113.32 of 4NucmMRespiratory chainNdi113.12 of 4NucmIMRespiratory chainQcr2133 of 4NucmMRespiratory chainNde1/Nde212.9/2.94 of 4NucmIMRespiratory chainMrp412.82 of 4NucmMProtein translation/stabilityYjl200c11.82 of 4NucmMCarbohydrate metabolismYjr039w10.93 of 4NucmNDNDGlt110.63 of 4NucmMaa and nitrogen metabolismSnq221.44 of 4Nucn*Pmal/Pma221.3/1.33 of 4Nucn*Mrh113.84 of 4Nucn*Ycl049c22.93 of 4NucNDYlr326w154 of 4NucNDYro212.62 of 4NucNDYkr078w11.94 of 4NucnDrs111.32 of 4NucnSas311.22 of 4Nucn Open table in a new tab The majority of the released peptides originates from proteins residing in the inner membrane (40%) and the matrix space (46%), whereas only one of these proteins has been localized to the intermembrane space or the outer membrane (Fig. 2B and Table I). These findings confirmed our conclusion that the proteolysis of nuclear-encoded mitochondrial proteins contributes to the peptide pool released from mitochondria and demonstrated that peptides are generated in different mitochondrial subcompartments. To control for the specificity of our procedure, we examined the temperature and ATP dependence of peptide accumulation in the mitochondrial supernatant. Mitochondria were incubated at 30 and 37 °C, and released peptides were analyzed by mass spectrometry as before. As expected from our pulse-labeling experiments (Fig. 1A), the number of detected peptides was slightly reduced after lowering the temperature to 30 °C (Fig. 2C). It should be noted, however, that to a large extent the same set of mitochondrial proteins was identified at both temperatures as a source of peptides, indicating that peptides are not predominantly derived from thermolabile mitochondrial proteins. Intramitochondrial ATP levels were reduced by pre-incubation of mitochondria with apyrase and oligomycin prior to shifting the samples to 37 °C. ATP depletion led to a drastically decreased number of detected peptides derived from mitochondrial proteins (Fig. 2D). This is in accordance with our previous studies demonstrating that the generation and export of peptides from mitochondria involves ATP-dependent proteolysis of mitochondrial proteins as well as the ABC transporter Mdl1 in the inner membrane (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). Characterization of Released Peptides—The large number of detected peptides demonstrates that a heterogeneous pool of peptides is released from mitochondria. Inspection of their amino acid sequences did not reveal any conserved features or sequence motifs. We noted a slight preference for charged, in particular negatively charged, versus hydrophobic amino acid residues but cannot exclude at present that this has technical reasons. The majority of the peptides identified is composed of 8–15 amino acid residues, which is consistent with our purification procedure (Fig. 3A). ∼50% of the released peptides originates from only three mitochondrial proteins (Table I), external NADH dehydrogenase (Nde1), cytochrome oxidase subunit 2 (Cox2), and Ylr356w, encoded by an uncharacterized open reading frame. Both Nde1 and Cox2 are anchored to the inner membrane and expose large domains to the intermembrane space, whereas Ylr356w most likely represents an integral membrane protein with four predicted membrane-spanning domains. The detected peptides cover large parts of the corresponding full-length protein, 54.3% of Nde1, 55.4% of Cox2, and 38.1% of Ylr356w (Fig. 3B). A similar sequence coverage was observed for some small mitochondrial proteins, like Stf1, Atp17, or Atp18 (Table I). In general, hydrophobic segments of membrane proteins were hardly represented by peptides detected in the mitochondrial supernatant (Fig. 3B). Their hydrophobicity may cause their preferential loss during the purification procedure. It is therefore conceivable that hydrophobic peptides are also released from mitochondria, resulting in an even higher sequence coverage in the case of multispanning membrane proteins. The majority of proteins, however, is only represented by a few or only one identified pep
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