Characterization of Peptides Released from Mitochondria

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. 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 substrates of other Moreover, the stability of the mitochondrial proteome and the extent of peptide export in yeast cells have not been In this we have therefore the proteolysis of newly and preexisting mitochondrial proteins and characterized peptides exported from mitochondria by mass Yeast and were at in on were of The has been K. Herrmann J.M. Stuart R.A. Mannhaupt G. Neupert W. Langer T. EMBO J. 1996; 15: 4218-4229Crossref PubMed Scopus (218) Google Scholar). of the and and were by a and a the gene a to allow the expression of a of in from were by a C. T. Biochem. PubMed Scopus Google Scholar), and in amino acid all amino acids two steps of mitochondria were in at a of for at to allow proteolysis to were into and by at for at The of the organelles by various mitochondrial mitochondria were to for at in and lacking and the mitochondria were in the of and were to at a peptide as (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). mitochondrial translation products were with in mitochondria in peptides composed of 6–20 amino acid residues were The of the were and in an Peptides in the were in of acid and by mass mitochondrial inner mitochondrial experiments that of the detected peptides under these conditions not were on a of mass were the system The a and a with were on a for acid at a rate of Peptides were the a of in acid at a rate of The and the were and established by the of to the of the of and by of the from to were for In the mass from to were generated from the the proteins were identified by the of the with a and mass of were and were peptide of a protein were identified and at of these peptides a were only in the set peptides were identified in at two of protein as mitochondrial to The is as demonstrated on the proteins at were as membrane proteins were localized to the intermembrane space of mitochondrial proteins on the functional by the C. H. K. M. C. T. 2004; PubMed Google Scholar). were from S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google of the of cells were at in with the and with for for mitochondrial protein by cells to with for at of into polypeptides by the of Mitochondria were as in and with of The mitochondrial in and into that were at the were at for at The in and of and within of to yeast cells growing logarithmically in at were to the were and cells by were by and by and and The peptide of for of in from and from an to the peptides exported from mitochondria, we first whether degradation products of nuclearly encoded mitochondrial proteins are extruded from the organelle. Yeast cells logarithmically growing on were for with Mitochondria were and at various to the release of from the organelle observed a and temperature-dependent of in the of the set free from mitochondria, this increased to at of released from mitochondria from cells on not In agreement with (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar), these proteolysis of mitochondrial from the of a acid the released from mitochondria is peptides and amino acid residues not of mitochondrial proteins under respiring and Yeast cells were at on on to the turnover of the mitochondrial cells were with to the stability of proteins newly imported into mitochondria, cells were for The release of degradation products from mitochondria at as in in the mitochondrial is as of in and the released from mitochondria were generated by the proteolytic breakdown of mitochondrial translation not in the upon of mitochondrial protein by the release of from mitochondria not by the of experiments revealed that mitochondrial protein under these conditions have that degradation products of mitochondrially encoded polypeptides only a of the released from the organelle. nuclearly encoded mitochondrial proteins are of the released of the by experiments in logarithmically growing yeast cells at of newly nuclearly as well as mitochondrially encoded proteins is by the release of the proteolytic breakdown products from the organelle. be by the efficiency of mitochondrial biogenesis in and of a number of the degradation of a of mitochondrial proteins be for the release of a of the from the organelle. these we the stability of the mitochondrial proteome by mitochondria were from logarithmically growing yeast cells and to a for at to allow proteolysis to In mitochondria were of yeast cells with to the stability of newly mitochondrial In agreement with A. J. C. R. B. A. B. P. C. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar), protein were identified on of protein we generated been at These experiments revealed a stability of the mitochondrial proteome and not allow the of protein that were an of the mitochondria at not therefore likely that the degradation of a of a number, a degradation of a number of for the observed proteolysis of mitochondrial the of Peptides from The of the of exported proteolytic breakdown products the of the peptides released from mitochondria. therefore peptide export experiments on a Mitochondria were from yeast cells logarithmically growing on and peptides in the were by a peptide with peptides of amino acid residues were and by into and only peptides identified in at two of were These experiments to the of 270 different peptides released from mitochondria that to 51 different proteins I and of these proteins has been localized to mitochondria by various the of has not been and of the identified peptide to Although of have been detected in mitochondria by mass spectrometry A. J. C. R. B. A. B. P. C. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar), most likely of of released of of of of of of of of of of of of of of of of of of of of of of of of of and of of of of of of of acid of of of of of of of of of of of of and of of of of of of of of of in a new The majority of the released peptides from proteins in the inner membrane and the matrix space only of these proteins has been localized to the intermembrane space the membrane and These that the proteolysis of mitochondrial proteins to the peptide released from mitochondria and demonstrated that peptides are generated in different mitochondrial control for the of we the and of peptide in the mitochondrial Mitochondria were at and and released peptides were by mass spectrometry as As from experiments the number of detected peptides the to be that to a extent the set of mitochondrial proteins identified at as a of indicating that peptides are not derived from mitochondrial were by of mitochondria with and to the to to a number of detected peptides derived from mitochondrial proteins This is in with studies that the and export of peptides from mitochondria 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). of number of detected peptides that a of peptides is released from mitochondria. of their amino acid not conserved a for in amino acid residues at that this has The majority of the peptides identified is composed of amino acid residues, is with of the released peptides from only mitochondrial proteins and encoded by an and are to the inner membrane and expose to the intermembrane most likely an membrane protein with The detected peptides of the of of and of observed for mitochondrial In of membrane proteins were represented by peptides detected in the mitochondrial their the is therefore that peptides are released from mitochondria, in an higher in the of membrane The majority of is only represented by a only identified Whereas different peptides were identified in different in peptides were in This is by the matrix protein Although is composed of amino acid residues, we detected the peptide to amino acids of in In other peptides of different to in the peptide were identified a of the i-AAA of peptides derived from their proteolysis within mitochondria. proteins are membrane are only poorly upon and have in the analysis of the mitochondrial proteome. therefore the stability of and within mitochondria. mitochondrial protein by in yeast cells on at membrane were by for the of and not a in the of protein within mitochondria other inner membrane proteins from peptides were to be released from mitochondria, remained under these conditions not These that only a of these proteins is degraded in logarithmically growing cells that be detected by subunits are substrates of the i-AAA protease (15Weber E.R. Hanekamp T. Thorsness P.E. Mol. Biol. Cell. 1996; 7: 307-317Crossref PubMed Scopus (119) Google Scholar, T. T. A. Mol. Cell. Biol. 15: PubMed Scopus Google Scholar, D.A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). whether the of peptides in the mitochondrial on proteolysis by the i-AAA protease, we the export of peptides from mitochondria lacking the i-AAA protease the ABC transporter Mdl1, proteins As the degradation of a number of peptides generated upon proteolysis of detected in the of mitochondria with and mitochondria peptides were only detected in of and mitochondria and were not set free from mitochondria This observation suggests that as is degraded by the i-AAA we of the observation that a to the of the protein in the is to the inner membrane and not The in cells the expression of in these the stability of at in and cells the protein by Although the degraded in it in cells lacking Yme1 from these experiments that the majority of and is in mitochondria. a of and is degraded by the i-AAA protease in the inner membrane, in the release of peptides from the intermembrane The predominant appearance of and peptides be partially by their within mitochondria. are the of for mitochondrial proteins S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The for mitochondrially encoded are not known at subunits are S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). we not a the protein within mitochondria, on and the of peptides in the mitochondrial peptides not products of the and under respiring conditions S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). in to other mitochondrial and expose to the intermembrane a that the export of peptides derived from these this as peptides derived from other intermembrane space are not represented in the peptide pool. Peptides from with in the of peptides released from mitochondria, the peptide were to functional by the C. H. K. M. C. T. 2004; PubMed Google Scholar). peptides originate from proteins with in protein translation and and to a extent in various other metabolic pathways of the identified proteins is associated with the respiratory chain is the of peptides derived from of membrane proteins the of this we the of known mitochondrial proteins in the the functional C. H. K. M. C. T. 2004; PubMed Google Scholar). in has only been to of these proteins of the mitochondrial proteins in mitochondrial translation protein of the identified peptide into this class therefore that the number of peptide with in the respiratory chain is not by the number of representatives of this functional in mitochondria. The of peptides released from mitochondria on the of the peptide within the organelle. of on and is by an increased of respiratory chain and acid S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, W. J. R.A. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). As analysis respiring the increased of respiratory chain the predominant appearance of peptides derived from proteins of this functional first for the expression of mitochondrial proteins can be from analysis of the yeast under various growth conditions S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, W. J. R.A. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). the number of for mitochondrial proteins in cells on S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google and their to the various functional Whereas only of for proteins with associated with the respiratory chain under respiring conditions, of the peptide into this the predominant appearance of peptides derived from proteins to the respiratory chain can only be partially by their increased expression under respiring growth of under the of peptides to oxidative we the stability of mitochondrial proteins under and respiring growth Yeast cells were in the of on as in mitochondria by the release of from the organelle. of the in the of mitochondria from respiring cells under these conditions of the mitochondrial proteome is degraded in logarithmically growing yeast cells of the the stability of proteins newly imported into mitochondria, a of yeast proteins with under and respiring conditions to mitochondria from cells on the of the from the mitochondrial cells were on we observed a increased proteolysis of newly imported proteins mitochondria from respiring cells of the in the mitochondrial under these from these experiments that the stability of newly mitochondrial proteins is under respiring growth In this we have demonstrated a constant efflux of peptides from mitochondria. These peptides are derived from a number of mitochondrial are localized in different subcompartments of the organelle. experiments have a turnover rate of the mitochondrial provided insight into the efficiency of mitochondrial and identified proteins newly imported into mitochondria as a for the exported The of a of peptides exported from mitochondria that peptide and thereby proteolysis of mitochondrial is in logarithmically growing yeast the the mitochondrial proteome is as revealed by not mitochondrial proteins a protein turnover this the analysis of peptide export from mitochondria of yeast cells to with sensitivity the stability of the mitochondrial proteome. observed the turnover of of the preexisting mitochondrial proteins under and respiring growth conditions, a to be detected in in Moreover, experiments new on the stability of proteins newly imported into mitochondria and thereby on the efficiency of protein biogenesis within the organelle. of mitochondrial proteins to be degraded into mitochondria in logarithmically growing yeast indicating that peptides exported from mitochondria are generated upon proteolysis of newly mitochondrial of newly imported proteins is most likely by impaired protein within mitochondria. The majority of the detected peptides from proteins in the inner membrane and the matrix space and is released from mitochondria in an ATP-dependent manner. This to the of ATP-dependent proteases in different subcompartments of mitochondria Dyck L. Langer T. Cell. Mol. Life Sci. 1999; 55: 825-842Crossref Scopus (86) Google Scholar). In agreement with peptides derived from mitochondrially encoded proteins were set free from mitochondria (10Young L. Leonhard K. Tatsuta T. Trowsdale J. Langer T. Science. 2001; 291: 2135-2138Crossref PubMed Scopus (178) Google Scholar). These polypeptides are degraded by the m-AAA protease in the of by the i-AAA protease (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, E.R. Hanekamp T. Thorsness P.E. Mol. Biol. Cell. 1996; 7: 307-317Crossref PubMed Scopus (119) Google Scholar, T. T. A. Mol. Cell. Biol. 15: PubMed Scopus Google Scholar, D.A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it is likely that nuclearly encoded inner membrane proteins are degraded by AAA proteases by the release of the proteolytic breakdown This is by is degraded by the i-AAA peptides released from mitochondria are generated upon degradation of a number of Although the m-AAA protease be in the proteolysis of proteins associated with the matrix of the inner membrane, as has been demonstrated for D. S. M. Langer T. EMBO 2004; PubMed Scopus Google Scholar), are likely substrates of the we detected peptides derived from a known of this protease D.A. Davies Cell Biol. 2002; PubMed Scopus Google Scholar). therefore that all ATP-dependent proteases in yeast mitochondria are in the of peptides that are released from mitochondria. Although a number of inner membrane and proteins has been identified as peptide inner membrane proteins are represented by a number of is the of peptides by This in protein that are to be detected in a proteome Moreover, oligopeptidases that have been identified within mitochondria A. J. C. R. B. A. B. P. C. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, A. M. H. in Cell Scholar, A. P. J. H. F. E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google degrade peptides generated by ATP-dependent proteases in the This peptides of different from of mitochondrial proteins were detected in the released peptide pool. this a of the ATP-dependent proteases functional of the proteins that have been identified as peptide revealed that proteins to oxidative are with the of mitochondrial proteins in different unlikely that this is a of their in the inner membrane, as a number of proteins has been identified the peptide a rate of proteolysis for all mitochondrial the of a protein in the appearance of peptides in the mitochondrial we not a the protein and the of of the most proteins are localized in the inner membrane intermembrane the of respiratory is not to the predominant of peptides are to the expression of mitochondrial proteins under respiring the appearance of peptides in the mitochondrial to be by in the of different functional that the biogenesis of the respiratory chain with efficiency with other mitochondrial In agreement with this the proteolysis of newly imported mitochondrial proteins is increased cells are growing under respiring on is known to to in gene mainly respiratory chain The of the within the mitochondrial is increased under respiring conditions S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, H. C. W. L. C. C. H. M. T. Biol. 2004; Scopus Google Scholar). the increased turnover of mitochondrial proteins under respiring conditions can at partially be by the increased of proteins to oxidative their increased proteolysis upon into mitochondria. This be by a oxidative of respiratory chain components and their proteolysis under respiring is in this that we not a of oxidative in peptide that proteins are not the for peptides released from mitochondria. The of in the mitochondrial and proteome has to the that the turnover of mitochondrial proteins under different physiological conditions S. U. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). results indeed that growth conditions can the stability of newly mitochondrial is therefore that the composition of the peptide exported from mitochondria in to a of peptides thereby the functional of mitochondria to the for with