The Energetics of Pex5p-mediated Peroxisomal Protein Import

Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system. Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system. Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally imported into the organelle. The vast majority of these proteins possess the so-called peroxisomal targeting sequence 1 (PTS1), 1The abbreviations used are: PTS, peroxisomal targeting sequence; PNS, post-nuclear supernatant; GST, glutathione S-transferase.1The abbreviations used are: PTS, peroxisomal targeting sequence; PNS, post-nuclear supernatant; GST, glutathione S-transferase. a C-terminal tripeptide complying to the consensus sequence S/A/C-K/R/H-L/M (1Gould S.G. Keller G A. Subramani S. J. Cell Biol. 1987; 105: 2923-2931Crossref PubMed Scopus (359) Google Scholar, 2Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (871) Google Scholar, 3Elgersma Y. Vos A. van den Berg M. van Roermund C.W. van der Sluijs P. Distel B. Tabak H.F. J. Biol. Chem. 1996; 271: 26375-26382Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Lametschwandtner G. Brocard C. Fransen M. Van Veldhoven P. Berger J. Hartig A. J. Biol. Chem. 1998; 273: 33635-33643Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). These PTS1-containing proteins are specifically targeted to the organelle matrix by Pex5p, the PTS1 receptor (5McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 6Van der Leij I. Franse M.M. Elgersma Y. Distel B. Tabak H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11782-11786Crossref PubMed Scopus (201) Google Scholar, 7Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (379) Google Scholar, 8Fransen M. Brees C. Baumgart E. Vanhooren J.C. Baes M. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 1995; 270: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar). According to current models of peroxisomal biogenesis, the Pex5p-mediated process of protein import can be divided into four steps. In the first step, newly synthesized PTS1-containing proteins interact with Pex5p in the cytosol. This protein-protein interaction involves the PTS1 signal on one side and the tetratricopeptide repeats domain of Pex5p on the other. The Pex5p-cargo protein complex is then recognized by the so-called docking machinery present in the peroxisomal membrane. Somewhere after this event, the PTS1-containing protein is released into the peroxisomal matrix. Finally, Pex5p is recycled back to the cytosol to catalyze additional rounds of transportation (reviewed in Refs. 10Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 69: 399-418Crossref PubMed Scopus (199) Google Scholar, 11Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar, 12Sparkes I.A. Baker A. Mol. Membr. Biol. 2002; 19: 171-185Crossref PubMed Scopus (34) Google Scholar). One obvious property of such cyclic mechanism is that it needs some form of energy input to function, and indeed, basically all the studies addressing this issue are unanimous in this respect: protein import into the peroxisomal matrix requires hydrolysis of ATP (13Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (150) Google Scholar, 14Behari R. Baker A. J. Biol. Chem. 1993; 268: 7315-7322Abstract Full Text PDF PubMed Google Scholar, 15Rapp S. Soto U. Just W.W. Exp. Cell Res. 1993; 205: 59-65Crossref PubMed Scopus (39) Google Scholar, 16Soto U. Pepperkok R. Ansorge W. Just W.W. Exp. Cell Res. 1993; 205: 66-75Crossref PubMed Scopus (46) Google Scholar, 17Wendland M. Subramani S. J. Cell Biol. 1993; 120: 675-685Crossref PubMed Scopus (103) Google Scholar, 18Horng J.T. Behari R. Burke L.E. Baker A. Eur. J. Biochem. 1995; 230: 157-163Crossref PubMed Scopus (25) Google Scholar, 19Brickner D.G. Harada J.J. Olsen L.J. Plant Physiol. 1997; 113: 1213-1221Crossref PubMed Scopus (24) Google Scholar, 20Pool M.R. Lopez-Huertas E. Baker A. EMBO J. 1998; 17: 6854-6862Crossref PubMed Scopus (18) Google Scholar, 21Terlecky S.R. Legakis J.E. Hueni S.E. Subramani S. Exp. Cell Res. 2001; 263: 98-106Crossref PubMed Scopus (24) Google Scholar). However, the precise step(s) of this import pathway where energy input is necessary has not been firmly established. For instance, it is generally accepted that the step of protein translocation across the peroxisomal membrane requires ATP hydrolysis. Such conclusion derives from the fact that ATP depletion or the inclusion of non-hydrolysable ATP analogues in the several experimental systems used result in an inhibition of the peroxisomal import process. However, the possibility that recycling of Pex5p back to the cytosol is an ATP-dependent event and the rate-limiting step in all this process was never considered. In this scenario, inhibition of peroxisomal protein import by lack of ATP would result from the non-availability of peroxisomal docking/translocation sites for the Pex5p-cargo protein complex and not from the inhibition of some ATP-dependent protein catalyzing the translocation of the cargo proteins across the organelle membrane. Furthermore, many newly synthesized proteins are bound by chaperones while still in the cytosol (or in a cell-free in vitro translation system). It is known that disruption of this kind of protein interaction requires ATP hydrolysis (reviewed in Ref. 22Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (917) Google Scholar). Thus, the observed ATP dependence of peroxisomal import may also be related, not to the translocation step per se (as assumed) but rather to the disruption of these chaperone-protein complexes with the concomitant production of import-competent cargo proteins. In fact, several data addressing the role of chaperones in peroxisomal protein import are already available and strongly suggest that this phenomenon does occur (23Walton P.A. Wendland M. Subramani S. Rachubinski R.A. Welch W.J. J. Cell Biol. 1994; 125: PubMed Scopus Google Scholar, W.J. Olsen L.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. R. N. Y. Biochem. J. 2001; PubMed Scopus Google Scholar). In this we the of peroxisomal protein import by the of ATP on the insertion of Pex5p into the peroxisomal membrane. data suggesting that insertion of Pex5p into the organelle membrane does not require ATP hydrolysis. This observation with the fact that of Pex5p from the organelle membrane is by C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google at the peroxisomal ATP is needed predominantly (if not exclusively) the recycling step of the PTS1 receptor. It is that protein translocation across the peroxisomal membrane is by protein-protein the peroxisomal targeting domain of Pex5p, on one and the peroxisomal docking/translocation machinery on the other. of for in post-nuclear for in vitro import C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of from by and was to Just W.W. A. Biochem. PubMed Scopus Google with C. C. C. J.E. Biochem. PubMed Scopus Google Scholar). in 1 in and at It is that this in some of the peroxisomal matrix a that the in the cargo dependence of Pex5p import is However, the for the protein complex is Gould S.J. Berg PubMed Scopus Google this of PTS1-containing proteins in import would be that of the to of peroxisomal from Ref. B. Lazarow P.B. C. J. Cell Biol. PubMed Scopus (103) Google is released into the the of the from the and additional and that the of with in of (13Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (150) Google was to peroxisomal (if at all In at for in of import and of a Pex5p and of or of In some import was used of import and by C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In inhibition of with of in of import for on the import by to of Ref. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a PTS1 signal at the Ref. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and with a sequence; Ref. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google proteins and used in import at of and of with was on for in import B. on for with 1 from a in the the of in the after the with import B. 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Full Text Full Text PDF PubMed Scopus Google Scholar). also that insertion of Pex5p into the peroxisomal membrane is strongly in the of non-hydrolysable ATP or by ATP depletion of the import C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This observation suggest that the peroxisomal import of Pex5p requires However, C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the possibility that the majority of the docking/translocation sites of the peroxisomal membrane already by Pex5p not be The fact that peroxisomal Pex5p is from the organelle in an ATP-dependent process C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google conclusion on the role of ATP (if on the Pex5p import an to the of import of Pex5p is by peroxisomal of Pex5p, we to in the For this a was in the of 1 ATP at for that peroxisomal of Pex5p Ref. C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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The of Pex5p in these peroxisomal to that of Pex5p from the membrane of for some to this in in vitro import from import Pex5p to an in vitro import However, the import of these is with the of a we need to in these import of to the of imported Pex5p with of a that peroxisomal proteins to of protein Just W.W. A. Biochem. PubMed Scopus Google this observation the import of at In fact, this is import of the in vitro synthesized Pex5p is not by Pex5p are used in these this peroxisomal import of Pex5p it is cargo and it requires available on the peroxisomal membrane. insertion of Pex5p into the peroxisomal membrane requires the of PTS1-containing proteins in the import C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This is not a in in vitro import the of peroxisomal PTS1-containing proteins released into the the of is to an import of phenomenon is observed are used in in vitro import insertion of Pex5p into the peroxisomal membrane can be PTS1-containing proteins to the import However, an inhibition on the import of Pex5p is observed the import is in the of a protein of Pex5p This of the PTS1 receptor the domain of Pex5p PTS1-containing proteins present in the import but peroxisomal targeting G. D. E. P. Gould S.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. N. Y. Mol. Biol. 2002; PubMed Scopus Google Scholar). of this can be by a PTS1-containing protein in the import Thus, these experimental insertion of Pex5p into the peroxisomal membrane is cargo is one of the of the docking/translocation complex (reviewed in Refs. 10Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 69: 399-418Crossref PubMed Scopus (199) Google Scholar, 11Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar, 12Sparkes I.A. Baker A. Mol. Membr. Biol. 2002; 19: 171-185Crossref PubMed Scopus (34) Google and to this peroxisomal import of Pex5p C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are with and then to an import a inhibition on the import process of Pex5p is observed data strongly suggest for the in the Pex5p-mediated import the of a in in vitro import However, the the systems not the to Pex5p Furthermore, in vitro imported Pex5p can be back to the of the import These from in vitro in are to a import In the first step, Pex5p is imported into for a of in the step, import of the PTS1 receptor is by a vast of to the the of the imported Pex5p a of is in Pex5p is imported into in the of ATP and then to a in the of the in the of Pex5p can be This result is in with that a is used in this of in vitro imported Pex5p is from the peroxisomal in an ATP-dependent process C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is also in the of Pex5p can be in these is to an import the are not to of Pex5p, suggesting that some necessary for the of Pex5p was or the of the the possibility to the of these by the import with cytosolic not of Pex5p into the of of that are not in Pex5p from the organelle membrane but are still to import the PTS1 we the of this process. For this and the to a ATP depletion import these One the ATP or to a of in on the peroxisomal import of Pex5p can be these experimental Most Thus, the for on the in vitro import of Pex5p was also of this from the import or the inclusion of the and in the import on the peroxisomal import of Pex5p These strongly suggest that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. The of peroxisomal protein import has from the of this experimental systems been used by several to this The unanimous conclusion that from these studies is that energy by ATP hydrolysis is necessary for peroxisomal protein import into the organelle matrix (13Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (150) Google Scholar, 14Behari R. Baker A. J. Biol. Chem. 1993; 268: 7315-7322Abstract Full Text PDF PubMed Google Scholar, 15Rapp S. Soto U. Just W.W. Exp. Cell Res. 1993; 205: 59-65Crossref PubMed Scopus (39) Google Scholar, 16Soto U. Pepperkok R. Ansorge W. Just W.W. Exp. Cell Res. 1993; 205: 66-75Crossref PubMed Scopus (46) Google Scholar, 17Wendland M. Subramani S. J. Cell Biol. 1993; 120: 675-685Crossref PubMed Scopus (103) Google Scholar, 18Horng J.T. Behari R. Burke L.E. Baker A. Eur. J. Biochem. 1995; 230: 157-163Crossref PubMed Scopus (25) Google Scholar, 19Brickner D.G. Harada J.J. Olsen L.J. Plant Physiol. 1997; 113: 1213-1221Crossref PubMed Scopus (24) Google Scholar, 20Pool M.R. Lopez-Huertas E. Baker A. EMBO J. 1998; 17: 6854-6862Crossref PubMed Scopus (18) Google Scholar, 21Terlecky S.R. Legakis J.E. Hueni S.E. Subramani S. Exp. Cell Res. 2001; 263: 98-106Crossref PubMed Scopus (24) Google Scholar). However, the exact in the Pex5p-mediated import pathway where energy input is necessary unclear. Here, we that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This with data that peroxisomal of the PTS1 receptor is by C. C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google also strongly suggest at the peroxisomal membrane ATP is predominantly (if not exclusively) used to reset the Pex5p-mediated transport to Pex5p back into the cytosol. This conclusion does not the need for ATP at of the peroxisomal protein import In fact, we that many peroxisomal proteins (if not are imported into the organelle in Rachubinski R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, J. Cell Biol. 1994; PubMed Scopus Google Scholar, P.A. P.E. Subramani S. Mol. Biol. 1995; PubMed Scopus Google Scholar, T. E. C. J. Cell Biol. 1996; PubMed Scopus Google Scholar, P.B. J. Cell Biol. 1996; PubMed Scopus Google Scholar, J.J. Rachubinski R.A. J. Cell Biol. 1998; PubMed Scopus Google Scholar, Rachubinski R.A. J. Cell Biol. 2002; PubMed Scopus (103) Google and that protein is a process that requires ATP hydrolysis (reviewed in Ref. 22Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (917) Google then ATP is used of the translocation It be that in the experimental used import of Pex5p into the peroxisomal membrane in the of already PTS1-containing proteins that from the the of the used in these Thus, this of the Pex5p-mediated import pathway can be from these of peroxisomal Pex5p present in several in PTS1 protein import to the that and known be in the of Pex5p back to the cytosol J.E. J.C. Gould S.J. Mol. Biol. 2000; PubMed Scopus Google Scholar). these data it was that and of the translocation step in the transport of proteins across the peroxisomal membrane in the of the docking/translocation machinery S.J. Nat. Rev. Mol. Biol. 2002; PubMed Scopus Google Scholar). the of the in the of Pex5p from the peroxisomal was the of this data strongly a role of and in of these Thus, and are in the process of protein translocation across the peroxisomal then the that these are in the of peroxisomal Pex5p that insertion of Pex5p into the peroxisomal membrane does not require hydrolysis of ATP raises an the of protein translocation across the peroxisomal membrane. ATP is in this step and a membrane is for this process (13Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (150) Google Scholar, 17Wendland M. Subramani S. J. Cell Biol. 1993; 120: 675-685Crossref PubMed Scopus (103) Google E. A. M. R. A. C. and J. E. then form of energy is used to transport proteins a the to this on the protein-protein that the PTS1 receptor with the docking/translocation machinery of the peroxisomal membrane. on the of this protein is still data the of and on the docking/translocation complex are C. C. J.E. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. W. M. Eur. J. Cell Biol. 2001; PubMed Scopus Google Scholar, B. P. A. W. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). of these membrane are of to the PTS1 receptor M. T. N. Y. Mol. Biol. 2002; PubMed Scopus Google Scholar, M. S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, Gould S.J. J. Cell Biol. 761-774Crossref PubMed Scopus Google Scholar, N. R. Y. S. T. S. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. X. G. R. Mol. Biol. 19: PubMed Scopus Google Scholar, I. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and in the for protein translocation across the peroxisomal membrane in of these not is known the of the Pex5p interaction with or The is and several of this interaction may be the of Pex5p sites for of these sites with in the W. J. B. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. G. A. W. W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is to suggest in at some of these sites are by C. C. J.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, interacts with the C-terminal domain M. Brees C. Mannaerts G.P. D. Van Veldhoven P.P. Mol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, C. W. C. J.E. 2002; PubMed Scopus (39) Google Scholar). Thus, in the interaction is The energy in the complex is not known at the on a the energy released the interaction of one with Pex5p would be to one protein from the cytosol to the peroxisomal matrix a such require membrane such an mechanism of protein translocation across a membrane would not be the translocation step of many protein complexes across the has been to be (reviewed in Ref. A. Annu. Rev. Genet. 2001; PubMed Scopus Google Scholar). The data suggesting that ATP is for protein translocation across the peroxisomal may in with the observation that Pex5p G. Gould S.J. J. Cell Biol. 1996; PubMed Scopus Google and newly synthesized PTS1-containing proteins E. 1987; Full Text PDF PubMed Scopus Google in at the peroxisomal However, all of these can be by that docking/translocation complex has at sites for Pex5p one for docking and one for the translocation of Pex5p to of these sites would to However, in the of energy the of available translocation sites would of Pex5p is to an of Pex5p-cargo protein complexes at the docking the protein in the peroxisomal docking step of the Pex5p-cargo protein complex is one of