Ribosome Binding to Mitochondria Is Regulated by GTP and the Transit Peptide

Abstract

The association between ribosomes and the pore proteins at the endoplasmic reticulum membrane is important to co-translational translocation. To determine if a similar association occurs between the ribosome and mitochondrial membrane protein(s) during protein import in higher eukaryotes, we examined ribosome-mitochondria binding. By using spectral measurements, analysis of mitochondrial associated RNA, and electron microscopy, we demonstrated that ribosomes stably bind to purified rat liver mitochondria in vitro. Binding of ribosomes to mitochondria was markedly reduced by GTP and nearly abolished by the non-hydrolyzable GTP analogue, guanosine-5′-[thio]-triphosphate (GTPγS), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for 92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTPγS did. These data show that receptors specific for ribosomes are present on the mitochondrial membrane, and a GTP-dependent process mediates this binding. The presence of a nascent chain alters these binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria. The association between ribosomes and the pore proteins at the endoplasmic reticulum membrane is important to co-translational translocation. To determine if a similar association occurs between the ribosome and mitochondrial membrane protein(s) during protein import in higher eukaryotes, we examined ribosome-mitochondria binding. By using spectral measurements, analysis of mitochondrial associated RNA, and electron microscopy, we demonstrated that ribosomes stably bind to purified rat liver mitochondria in vitro. Binding of ribosomes to mitochondria was markedly reduced by GTP and nearly abolished by the non-hydrolyzable GTP analogue, guanosine-5′-[thio]-triphosphate (GTPγS), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for 92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTPγS did. These data show that receptors specific for ribosomes are present on the mitochondrial membrane, and a GTP-dependent process mediates this binding. The presence of a nascent chain alters these binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria. Proteins can cross membranes by either post-translational or co-translational translocation. Based on in vitroobservations, proteins targeted to the mitochondria are thought to be completely synthesized in the cytoplasm and cross the mitochondrial membrane(s) post-translationally (1Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (920) Google Scholar). However, evidence consistent with a co-translational translocation pathway for mitochondrial protein import has been reported. For example, 1) the surface of mitochondria isolated from cycloheximide-treated yeast cells is observed to be studded with polysomes (2Kellems R. Butow R. J. Biol. Chem. 1972; 247: 8043-8050Abstract Full Text PDF PubMed Google Scholar); 2) the number of bound polysomes is dependent on the metabolic state of the cells from which the mitochondria are isolated (3Kellems R. Butow R. J. Biol. Chem. 1974; 249: 3304-3310Abstract Full Text PDF PubMed Google Scholar); 3) ribosomes are bound to the mitochondria at the contact sites (4Kellems R. Allison V. Butow R. J. Cell. Biol. 1975; 65: 1-14Crossref PubMed Scopus (182) Google Scholar); and 4) the mRNA of polysomes that co-isolate with mitochondria is enriched in messages for mitochondrial proteins (5Ades I. Butow R. J. Biol. Chem. 1980; 255: 9918-9924Abstract Full Text PDF PubMed Google Scholar, 6Suissa M. Schatz G. J. Biol. Chem. 1982; 257: 13048-13055Abstract Full Text PDF PubMed Google Scholar). In addition, both in vitroand in vivo, mitochondrial protein import can be instantaneously disrupted by cycloheximide-induced translation arrest indicating that no detectable pool of full-length precursor exists in the cytosol and that translation and import are tightly coupled (7Verner K. Trends Biochem. Sci. 1993; 18: 366-371Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 8Fujiki M. Verner K. J. Biol. Chem. 1991; 266: 6841-6847Abstract Full Text PDF PubMed Google Scholar, 9Fujiki M. Verner K. J. Biol. Chem. 1993; 268: 1914-1920Abstract Full Text PDF PubMed Google Scholar). Furthermore, methotrexate, which inhibits post-translational import of cytochrome oxidase subunit IV-dihydrofolate reductase by preventing its unfolding, does not inhibit cytochrome oxidase subunit IV-dihydrofolate reductase import in vivo (9Fujiki M. Verner K. J. Biol. Chem. 1993; 268: 1914-1920Abstract Full Text PDF PubMed Google Scholar). This indicates that co-translational translocation may eliminate the need for precursor proteins to be maintained in a translocation-competent state by chaperones in the cytoplasm. Almost all mitochondrial matrix and inner membrane proteins are synthesized with an N-terminal presequence and then imported into the mitochondria (10Mayer A. Neupert W. Lill R. Cell. 1995; 80: 127-137Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 11Roise D. Schatz G. J. Biol. Chem. 1988; 263: 4509-4511Abstract Full Text PDF PubMed Google Scholar, 12Kiebler M. Becker K. Pfanner N. Neupert W. J. Membr. Biol. 1993; 135: 191-207Crossref PubMed Scopus (82) Google Scholar). The presequence-containing precursor protein is presumably held in a translocation-competent conformation in the cytoplasm by hsp70 chaperonins probably in concert with the presequence-specific chaperonin, e.g. “targeting factor” or mitochondrial stimulating factor, and the precursor is recognized and imported by a multisubunit translocation complex in the mitochondrial membranes (13Deshaies R.J. Koch B.D. Werner-Washburne M. Craig A.E. Schekman R. Nature. 1988; 333: 800-805Crossref Scopus (1003) Google Scholar, 14Murakami H. Pain D. Blobel G. J. Cell Biol. 1988; 107: 2051-2057Crossref PubMed Scopus (279) Google Scholar, 15Ono H. Tuboi S. Arch. Biochem. Biophys. 1990; 280: 299-304Crossref PubMed Scopus (33) Google Scholar, 16Hachiya N. Komiya T. Alam R. Iwahashi J. Sakaguchi M. Omura T. Mihara K. EMBO J. 1994; 13: 5146-5154Crossref PubMed Scopus (116) Google Scholar, 17Hachiya N. Mihara K. Suda K. Horst M. Schatz G. Lithgow T. Nature. 1995; 376: 705-709Crossref PubMed Scopus (156) Google Scholar, 18Komiya T. Mihara K. J. Biol. Chem. 1996; 271: 22105-22110Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Since these early events in targeting and translocation clearly and specifically involve the N-terminal presequence, it is entirely possible that mitochondrial protein import can be initiated long before translation is complete. To examine the hypothesis that, in vivo, proteins import into the mitochondria by a co-translational translocation pathway, and to determine whether receptor(s) for the translation machinery at the outer membrane play a role in targeting an incompletely synthesized nascent polypeptide chain to the mitochondrial import site, we examined the interaction of the ribosome with the mitochondria. Specifically, ribosome binding to mitochondria was shown using three independent methods, and the ability of ribosome-bound nascent polypeptide chains to target the ribosome to the mitochondria was determined. Finally, because recent studies have shown that GTP-binding proteins are not only involved in initiating protein translocation at the membrane of the endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; RER, rough endoplasmic reticulum; GTPγS, guanosine-5′-[thio]-triphosphate; EKMT, EDTA, high salt-washed mitochondria; EKMPMT, EDTA/high salt, metrizamide/Percoll-purified mitochondria; mMDH, mitochondrial malate dehydrogenase; SRP, signal recognition particle; GNRP, guanine nucleotide release or exchange protein. but are also involved in protein import into chloroplast (19Kessler F. Blobel G. Patel H.A. Schnell D.J. Science. 1994; 266: 1035-1039Crossref PubMed Scopus (245) Google Scholar), we determined the effect of ATP and GTP on ribosomes binding to mitochondria and the early events in protein translocation. Ribosomes and rough endoplasmic reticulum (RER) were isolated from the post-mitochondrial supernatant of rat liver homogenates by a previously described method (20Bloemendel H. Benedetti E.L. Bont W.S. Methods Enzymol. 1974; 30: 313-327Crossref PubMed Scopus (22) Google Scholar). To remove endogenous GTPase activity, isolated ribosomes were resuspended in 50 mm Tris, pH 7.4, 0.5 m KOAc, 10 mmMgCl2, and 4 mm dithiothreitol containing 1 unit/μl RNasin (Promega), incubated at room temperature 15 min, and re-isolated by sedimentation through a 0.5 m sucrose cushion containing 0.5 m KOAc, 50 mm Tris, pH 7.6, 5 mm MgOAc, 4 mm dithiothreitol, 1 unit/μl RNasin. Rabbit reticulocyte ribosomes were isolated as described (21Lauring B. Kreibich G. Wiedmann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 9435-9439Crossref Scopus (71) Google Scholar). The final ribosomal pellets were resuspended in ribosome binding buffer (RB, 50 mm Tris, pH 7.4, 100 mm KOAc, 10 mm MgCl2, 1 mm dithiothreitol containing 1 unit/μl RNasin) and stored at −70 °C in small aliquots. Mitochondria were isolated from Sprague-Dawley rat livers using previously published methods (22Chu T. Grant P. Strauss A. J. Biol. Chem. 1987; 262: 12806-12811Abstract Full Text PDF PubMed Google Scholar). Mitochondria were further purified to reduce ER contamination using metrizamide-Percoll density gradient as described previously and Methods Scholar). were to a protein between 5 or 15 in 10 mm Tris, pH 7.4, mm and mm sucrose and either used or in and stored at −70 binding and GTP hydrolysis was the for and previously mitochondria, electron of the was disrupted in the previously mitochondria. Mitochondria were using a previously described method for ER membranes of ribosomes P. Blobel G. Methods Enzymol. PubMed Scopus Google Scholar). For all EDTA, high salt-washed mitochondria were used and were as described previously J. P. P. R. 1995; Scholar, G. Biochem. J. PubMed Scopus Google Scholar, The of from hydrolysis by mitochondria and ribosomes was determined using a of a previously described method D. Methods Enzymol. 1974; 30: PubMed Scopus Google Scholar). were as described and and the were to with and 100 of 100 of and of were a the was at or To for nucleotide containing ribosomes, mitochondria, or ribosome-mitochondria but as as containing only were in and was from the mitochondrial malate dehydrogenase mRNA was synthesized of as described previously (22Chu T. Grant P. Strauss A. J. Biol. Chem. 1987; 262: 12806-12811Abstract Full Text PDF PubMed Google Scholar). were in the and by in were in reticulocyte in the presence of to chain of P. A.E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The was in the and chain were using the reticulocyte as by the chain were isolated as described (21Lauring B. Kreibich G. Wiedmann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 9435-9439Crossref Scopus (71) Google the ribosomes were not high and the sucrose cushion 100 of mm binding ribosomes were at for to remove Mitochondria as and ribosomes as were in a of or of GTPγS, or was to a final of mm as were incubated for at room and mitochondria were at for The mitochondrial pellets were with and resuspended in containing 1 mmMgCl2, and the of the supernatant mitochondrial was at and for In containing only mitochondria and nucleotide were and as for the mitochondria, and nucleotide ribosomes nascent the of chain complex binding was also determined by of the To that of the resuspended mitochondrial pellets ribosomal of mitochondria were incubated with of ribosomes in of for 5 at room The ribosome-mitochondria were into 5 min, 4 and resuspended in of containing either or 5 mm GTPγS, or no were incubated at room temperature for 5 and through an of m sucrose cushion 5 min, 4 and were with and the The resuspended was to both and using a that been by chain using as a number The was by analysis to that the by to and not mitochondrial were for electron by 100 of mitochondria or ribosome-mitochondria of mitochondrial to 1 of containing 100 100 mm pH were with 100 mm 100 mm pH 7.4, in and in were in J. PubMed Scopus Google Scholar), and examined using a at of for mitochondria were at a of and the number of ribosomes was by To that the of mitochondrial surface was used to the surface of of mitochondria PubMed Scopus Google Scholar). all in all this was at to and of ribosomes are for between For the with the and of ribosomes were and the and of the are reported. liver mitochondria by have been shown to a of rough endoplasmic G. Biochem. J. PubMed Scopus Google Scholar). In to examine only the ribosome-mitochondria mitochondrial were either to and high P. Blobel G. Methods Enzymol. PubMed Scopus Google Scholar), by density gradient and Methods Scholar), or were for of the and Based on activity, either density gradient or EDTA/high of mitochondria reduced the of contamination by whereas density gradient by EDTA/high contamination to ribosome-mitochondria binding rat liver ribosomes were in high to remove endogenous GTPase and were for from GTP or The initial rate of GTP hydrolysis by ribosomes was reduced by high Ribosomes were also for and were shown to be of contamination were for incubated at room temperature for 10 salt-washed salt-washed ribosomes salt-washed ribosomes salt-washed ribosomes mitochondria salt-washed ribosomes salt-washed ribosomes Mitochondria salt-washed ribosomes salt-washed ribosomes mitochondria the from at three of EDTA/high salt-washed mitochondria, 5 of ribosomes, and mm were for incubated at room temperature for 10 in a the from at three of EDTA/high salt-washed mitochondria, 5 of ribosomes, and mm salt-washed ribosomes were incubated with EDTA/high density gradient purified mitochondria the ribosome-mitochondria were from ribosomes by and the of resuspended the ribosomes in the supernatant were The in the between resuspended mitochondria incubated ribosomes and mitochondria incubated with ribosomes was to ribosome binding. shown in ribosomes bind to both isolated and previously mitochondria. The number of ribosomes bound of mitochondria is higher in the previously presumably because the mitochondrial of the mitochondria isolated mitochondria bind ribosome of mitochondrial protein with ribosomes ATP is for post-translational import of proteins into the mitochondria and because GTP is for co-translational translocation of protein into the ER, we that ribosome binding to mitochondria. of ribosome-mitochondria was as before were incubated with or GTPγS, or CTP. in the in an was observed resuspended mitochondria been incubated with ribosomes to resuspended mitochondria been incubated This indicates that the ribosomes with mitochondria, and the in was to ribosome binding in the were observed mm was in binding However, mm GTP or GTPγS was only in were observed of GDP or ATP in the binding in that were in the of nucleotide but were also higher GTP and This indicates that the association between ribosomes and mitochondria can be by GTP or GTPγS but is only by ATP or the used it is if the effect of ATP on binding is or a of of ATP to GTP from However, the of GTP or GTPγS on complex was from complex all indicating that GTP a role in the ribosome-mitochondria To that the in the and the for mitochondria incubated with ribosomes were to ribosome-mitochondria ribosome-mitochondria were by of with high salt-washed ribosomes in the of The were isolated by and the pellets were resuspended in containing either no GTPγS, or ATP at a of either or 5 were re-isolated by and to whether ribosomes bound the of in the and pellets was determined by the to by or by analysis from the the stably bound ribosomes, whereas in the supernatant ribosomes that were from the mitochondria. no nucleotide was to the nearly all with the mitochondria this indicates that the ribosomes stably bound to mitochondria in the of pellets from ribosome-mitochondria incubated with or GTPγS that guanine binding 4 ATP not binding these and the effect of GDP on binding was These were observed at of GTP not containing mitochondria were as the ribosome-mitochondria and to analysis in but no was detectable for these In with spectral GTP and GTPγS have the effect on complex These data show that 1) the with in ribosome and 2) the interaction is not the of binding ribosome-mitochondria are this interaction can be specifically by GTP or Finally, to that the spectral data and analysis ribosome-mitochondria binding and not as ribosome and ribosome-mitochondria were examined by electron mitochondria were and in was and in The mitochondria were then EDTA/high salt-washed and an of these mitochondria was and Ribosomes were then to the of the and the was incubated for 5 at room was then and the ribosome-mitochondria were in and as The was into and GTP was to of the at a final of 1 These were incubated for 5 min, in and then shown in 5 ribosomes are bound to the mitochondrial membrane surface to EDTA/high salt-washed mitochondrial membranes are of ribosomes 5 but ribosomes are to these mitochondria, ribosomes bind to the mitochondrial membrane surface 5 Finally, GTP is to the ribosome-mitochondria the number of ribosomes associated with the mitochondrial surface is markedly reduced with ribosome-mitochondria before of GTP 5 and or with ribosome-mitochondria incubated GTP not To these observed in ribosome we the number of ribosomes binding to mitochondria from 10 of all 5 that the observed in ribosome binding from 5 is and that is a between these data show that ribosomes with mitochondria bound to the mitochondrial surface but that these ribosomes can be completely by EDTA/high EDTA/high however, does not mitochondria to ability to bind ribosomes Finally, GTP is at ribosomes from the mitochondrial surface and clearly the interaction In we have used three to that ribosomes specifically bind to mitochondria. These data also show that the interaction is by GTP and is evidence that this interaction is the presence of nucleotide a effect on ribosome-mitochondria we that nucleotide hydrolysis was associated with this binding. The of on the of isolated mitochondria was in the and presence of ribosomes and sedimentation of the mitochondria, no was in the from EKMT, high salt-washed ribosomes, or ribosomes and incubated a In addition, was EKMT, ribosomes, or ribosomes and were incubated with GTPγS In the for from GTP by EKMPMT, in the and presence of The initial rate of hydrolysis of GTP by mitochondria was but was higher at for mitochondria and ribosomes Since ribosomes no GTPase activity, of by mitochondria in the presence of ribosomes is to the interaction between mitochondria and ribosomes the rate of GTP The rate of in the presence of ATP the rate of ATP hydrolysis by mitochondria in the presence of ribosomes, is to the of the of by mitochondria and ribosomes indicating that the ribosome-mitochondria interaction does not the rate of ATP hydrolysis the interaction GTP hydrolysis which this is a specific interaction that is by a GTP-dependent The presence of sites on the mitochondria that binding be important in co-translational translocation. of however, ribosomes isolated from the post-mitochondrial cytosol not stably bind to mitochondria. Since the is for targeting a mitochondrial protein in the post-translational import pathway, we that a nascent chain containing a mitochondrial be of the ribosome to the mitochondria and binding in the presence of whereas ribosomes nascent chains unprogrammed ribosomes in the presence of Ribosomes of the amino acids of the polypeptide chain from of the cytosol but of the N-terminal of the nascent polypeptide to the cytosol to translation W.S. A. Biochem. Biol. PubMed Scopus Google Scholar). mRNA a and containing the of the N-terminal of precursor was to of the of of the to the cytosol J. Strauss A. PubMed Scopus Google Scholar). The mRNA for this polypeptide was using in reticulocyte and the chain were isolated by and the were for binding in the or presence of or nascent polypeptide was used as a targeted protein and was to the The of these are in and show that the ribosomes a mitochondrial nascent the presence of GTP does not reduce the of chain bound to the mitochondria with the of chain bound no nucleotide is present GDP was the number of chain bound to mitochondria was not from the number of bound in the of However, the presence of GTPγS reduced the of ribosome-mitochondria These from findings using unprogrammed ribosomes that that GTP a in ribosome-mitochondria complex In addition, the ribosomes the nascent chains unprogrammed ribosomes and not stably bind to mitochondria in the presence of either GTP or the presence of the GTP-dependent release of the ribosome from the mitochondria and of a These show that the of ribosome-mitochondria binding in the presence of GTP and a for import in the presence of a ribosome unprogrammed ribosomes and ribosomes programmed with proteins are not stably bound to the mitochondrial it is that in the presence of ribosomes, the is not of the nascent polypeptide to the mitochondria the non-hydrolyzable GTPγS this binding. GTP-dependent in ribosome binding to mitochondria. In this we have demonstrated that ribosomes bind specifically to rat liver mitochondria and that both GTP and the of the nascent chain to the ribosome binding. Specifically, GTP ribosome-mitochondria binding mitochondrial translation were with the In addition, a of GTP completely abolished the of ribosome-mitochondria whether or not ribosomes presequence-containing nascent also demonstrated that whereas isolated mitochondria hydrolysis of GTP was ribosomes were to the This but does not the hypothesis of co-translational mitochondrial import by a in this ribosomes and stably bind to mitochondria. data that the interaction of ribosomes with the mitochondria and by a pathway, similar to used to co-translational translocation at the ER is that ribosomes target to the translocation the complex at the ER membrane independent of the signal recognition (21Lauring B. Kreibich G. Wiedmann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 9435-9439Crossref Scopus (71) Google Scholar, A. M. B. K. T. Biol. Cell. PubMed Scopus Google Scholar). The interaction of the ribosome with both and the translocation at the ER membrane GTP in both G. H. B. T. Dobberstein B. Nature. 1996; PubMed Scopus Google Scholar). the ER membrane, GTP also the release of from and the of of the chain complex to the In ribosomes, the ability to bind specifically to the ER but the between of the targeting the the and the to protein mitochondrial chaperonin, mitochondrial stimulating factor, has been with however, it is an protein and has only been shown to post-translationally no interaction with the ribosome (13Deshaies R.J. Koch B.D. Werner-Washburne M. Craig A.E. Schekman R. Nature. 1988; 333: 800-805Crossref Scopus (1003) Google Scholar, 14Murakami H. Pain D. Blobel G. J. Cell Biol. 1988; 107: 2051-2057Crossref PubMed Scopus (279) Google Scholar, 15Ono H. Tuboi S. Arch. Biochem. Biophys. 1990; 280: 299-304Crossref PubMed Scopus (33) Google Scholar, 16Hachiya N. Komiya T. Alam R. Iwahashi J. Sakaguchi M. Omura T. Mihara K. EMBO J. 1994; 13: 5146-5154Crossref PubMed Scopus (116) Google Scholar). In ribosome-mitochondria binding was only modestly by and the ribosome-mitochondria interaction not have effect on the rate of ATP ATP has a effect on binding or a for GTP is is however, is that GTPγS completely disrupted the ribosome-mitochondria binding that GTP hydrolysis has an important role in this that, similar to occurs at the ER membrane, ribosomes bind tightly to the mitochondrial membrane surface and that at GTP-binding factor, and the of the nascent chain bound to the the of this These to protein targeting to the ER have to that higher mitochondria ribosome similar to the which are in initiating co-translational translocation. observed that ribosome-mitochondria binding is by GTP the ribosome a nascent binding is in the of nucleotide and only by GDP indicates that ribosomes with mitochondria a of the binding is of nucleotide or with Since tightly bind ribosomes probably bind to mitochondria a of the binding is In it is the release of GDP by the of guanine nucleotide release or exchange protein that to bind GTP and the of a that hydrolysis of GTP by M. J. B. J. 1996; PubMed Scopus Google Scholar). GTPγS ribosome-mitochondria we that hydrolysis of GTP is not to binding. This that it is the of a ribosome-mitochondria binding that the Since both of and protein in GTP this is consistent with the that the rate of GTP hydrolysis by mitochondria in the presence of we that the ribosome-mitochondria interaction activity, and that GTP binding to an binding a is also that a is either on the or the mitochondria, and that the is on the that GTP binding the of the ribosome for the mitochondria. The ribosome-mitochondria interaction to the interaction that the of an that the occurs at the mitochondrial These data clearly show a between the translation machinery and the mitochondria, as as a ribosomal on mitochondrial GTP These findings are of the hypothesis that in vivo mitochondrial proteins are imported by co-translational translocation and the of GTP in the mitochondrial protein import (7Verner K. Trends Biochem. Sci. 1993; 18: 366-371Abstract Full Text PDF PubMed Scopus (98) Google Scholar). The is that precursor proteins are in the by chaperones to the mitochondrial and imported by a import However, the on which this is is for nearly all of the studies mitochondrial protein import have used in in which the precursor proteins are and then the is to mitochondria for This in the import but for co-translational import are completely by this of the protein import has been in yeast However, are in protein at the ER between yeast and higher that yeast is not of complex as in mitochondria Schekman R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, it is that the of a is that proteins not but are in specific R. J. Biochem. Cell Biol. 1996; PubMed Scopus Google Scholar). The that precursor proteins are in the cytosol and then target by is with the of a cytosol and be for cells with high of at completely synthesized mitochondrial proteins import post-translationally into isolated mitochondria, the that mitochondrial matrix and inner membrane proteins an N-terminal that long before protein is complete. is to the between mitochondria, ribosomes, the nascent and is however, that have in the membrane and contact sites of mitochondria of higher T. M. P. N. Biochem. Biophys. 1991; PubMed Scopus Google Scholar, S. K. S. K. J. Biochem. 1993; PubMed Scopus Google Scholar, M. M. P. Biophys. 1995; Scopus Google Scholar), and it has been that GTP hydrolysis is for post-translational protein import into yeast mitochondria N. N. Pain D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and to be and for of this and G. for in electron