Mapping the Cargo Protein Membrane Translocation Step into the PEX5 Cycling Pathway

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor. Over the last few years, valuable data on the mechanism of this process have been obtained using a PEX5-centered in vitro system. The data gathered until now suggest that cytosolic PEX5·cargo protein complexes dock at the peroxisomal docking/translocation machinery, where PEX5 becomes subsequently inserted in an ATP-independent manner. This PEX5 species is then monoubiquitinated at a conserved cysteine residue, a mandatory modification for the next step of the pathway, the ATP-dependent dislocation of the ubiquitin-PEX5 conjugate back into the cytosol. Finally, the ubiquitin moiety is removed, yielding free PEX5. Despite its usefulness, there are many unsolved mechanistic aspects that cannot be addressed with this in vitro system and that call for a cargo protein-centered perspective instead. Here we describe a robust peroxisomal in vitro import system that provides this perspective. The data obtained with it suggest that translocation of a cargo protein across the peroxisomal membrane, including its release into the organelle matrix, occurs prior to PEX5 ubiquitination. Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor. Over the last few years, valuable data on the mechanism of this process have been obtained using a PEX5-centered in vitro system. The data gathered until now suggest that cytosolic PEX5·cargo protein complexes dock at the peroxisomal docking/translocation machinery, where PEX5 becomes subsequently inserted in an ATP-independent manner. This PEX5 species is then monoubiquitinated at a conserved cysteine residue, a mandatory modification for the next step of the pathway, the ATP-dependent dislocation of the ubiquitin-PEX5 conjugate back into the cytosol. Finally, the ubiquitin moiety is removed, yielding free PEX5. Despite its usefulness, there are many unsolved mechanistic aspects that cannot be addressed with this in vitro system and that call for a cargo protein-centered perspective instead. Here we describe a robust peroxisomal in vitro import system that provides this perspective. The data obtained with it suggest that translocation of a cargo protein across the peroxisomal membrane, including its release into the organelle matrix, occurs prior to PEX5 ubiquitination. Peroxisomal matrix proteins are synthesized in cytosolic ribosomes and post-translationally targeted to the organelle (1Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (288) Google Scholar, 2Brown L.A. Baker A. Mol. Membr. Biol. 2008; 25: 363-375Crossref PubMed Scopus (67) Google Scholar). The vast majority of proteins destined to this compartment possess the so-called peroxisomal targeting sequence type 1 (PTS1), 3The abbreviations used are: PTS1peroxisomal targeting sequence type 1PTS2peroxisomal targeting sequence type 2DTMdocking/translocation machineryUbubiquitinUb·PEX5monoubiquitinated PEX5 speciesGST·Ubglutathione S-transferase-ubiquitinPNSpostnuclear supernatantGSHglutathioneATPγSadenosine 5-O-(thiotriphosphate)MOPS4-morpholinepropanesulfonic acid. a short domain present at their extreme C termini and frequently ending with the sequence SKL (3Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (934) Google Scholar, 4Brocard C. Hartig A. Biochim. Biophys. Acta. 2006; 1763: 1565-1573Crossref PubMed Scopus (219) Google Scholar). A small number of matrix proteins lack this domain and contain instead a PTS2, a degenerated nonapeptide with the sequence (R/K)(L/V/I)X5(H/Q)(L/A) present at their N termini (5Swinkels B.W. Gould S.J. Bodnar A.G. Rachubinski R.A. Subramani S. EMBO J. 1991; 10: 3255-3262Crossref PubMed Scopus (521) Google Scholar, 6Lazarow P.B. Biochim. Biophys. Acta. 2006; 1763: 1599-1604Crossref PubMed Scopus (116) Google Scholar). In contrast to the PTS1, which is not cleaved upon peroxisomal import, the PTS2 signal is proteolytically removed in the peroxisomal matrix of many organisms by a peroxisomal processing peptidase (1Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (288) Google Scholar, 7Kurochkin I.V. Mizuno Y. Konagaya A. Sakaki Y. Schönbach C. Okazaki Y. EMBO J. 2007; 26: 835-845Crossref PubMed Scopus (83) Google Scholar, 8Helm M. Lück C. Prestele J. Hierl G. Huesgen P.F. Fröhlich T. Arnold G.J. Adamska I. Görg A. Lottspeich F. Gietl C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11501-11506Crossref PubMed Scopus (77) Google Scholar). peroxisomal targeting sequence type 1 peroxisomal targeting sequence type 2 docking/translocation machinery ubiquitin monoubiquitinated PEX5 species glutathione S-transferase-ubiquitin postnuclear supernatant glutathione adenosine 5-O-(thiotriphosphate) 4-morpholinepropanesulfonic acid. In mammals and many other organisms, both PTS1-containing and PTS2-containing proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor (9Braverman N. Dodt G. Gould S.J. Valle D. Hum. Mol. Genet. 1998; 7: 1195-1205Crossref PubMed Scopus (158) Google Scholar, 10Otera H. Okumoto K. Tateishi K. Ikoma Y. Matsuda E. Nishimura M. Tsukamoto T. Osumi T. Ohashi K. Higuchi O. Fujiki Y. Mol. Cell. Biol. 1998; 18: 388-399Crossref PubMed Google Scholar, 11Woodward A.W. Bartel B. Mol. Biol. Cell. 2005; 16: 573-583Crossref PubMed Scopus (126) Google Scholar, 12Galland N. Demeure F. Hannaert V. Verplaetse E. Vertommen D. Van der Smissen P. Courtoy P.J. Michels P.A. Biochim. Biophys. Acta. 2007; 1773: 521-535Crossref PubMed Scopus (57) Google Scholar). PTS1 proteins interact directly with the C-terminal half of PEX5, a region comprising seven tetratricopeptide repeats arranged into a ring-like structure, whereas the PEX5-PTS2 interaction is bridged by the adaptor protein PEX7 (13Gatto Jr., G.J. Geisbrecht B.V. Gould S.J. Berg J.M. Proteins. 2000; 38: 241-246Crossref PubMed Scopus (30) Google Scholar, 14Brocard C. Kragler F. Simon M.M. Schuster T. Hartig A. Biochem. Biophys. Res. Commun. 1994; 204: 1016-1022Crossref PubMed Scopus (128) Google Scholar, 15Dodt 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 (390) Google Scholar, 16Matsumura T. Otera H. Fujiki Y. J. Biol. Chem. 2000; 275: 21715-21721Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17Dodt G. Warren D. Becker E. Rehling P. Gould S.J. J. Biol. Chem. 2001; 276: 41769-41781Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (178) Google Scholar, 19Williams C. Distel B. Biochim. Biophys. Acta. 2006; 1763: 1585-1591Crossref PubMed Scopus (46) Google Scholar). This adaptor protein interacts with a small region within the largely unfolded N-terminal half of PEX5 (17Dodt G. Warren D. Becker E. Rehling P. Gould S.J. J. Biol. Chem. 2001; 276: 41769-41781Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (178) Google Scholar, 20Carvalho A.F. Costa-Rodrigues J. Correia I. Costa Pessoa J. Faria T.Q. Martins C.L. Fransen M. Sá-Miranda C. Azevedo J.E. J. Mol. Biol. 2006; 356: 864-875Crossref PubMed Scopus (70) Google Scholar). Interestingly, not all proteins derived from the mammalian PEX5 gene have the capacity to bind PEX7. This is due to alternative splicing of the PEX5 transcript yielding two major mRNAs, one encoding the so-called large isoform of PEX5 (PEX5L) and the other coding for the small PEX5 isoform (PEX5S). PEX5S lacks a 37-amino-acid region that is involved in the PEX7 interaction, and so it is incompetent in the peroxisomal targeting of PTS2 proteins (16Matsumura T. Otera H. Fujiki Y. J. Biol. Chem. 2000; 275: 21715-21721Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17Dodt G. Warren D. Becker E. Rehling P. Gould S.J. J. Biol. Chem. 2001; 276: 41769-41781Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (178) Google Scholar). In recent years, valuable data on the mechanistic details of the PEX5-mediated protein import pathway in mammals have been obtained using a PEX5-centered in vitro system (21Miyata N. Fujiki Y. Mol. Cell. Biol. 2005; 25: 10822-10832Crossref PubMed Scopus (175) Google Scholar, 22Gouveia A.M. Guimaraes C.P. Oliveira M.E. Reguenga C. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 226-232Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 23Grou C.P. Carvalho A.F. Pinto M.P. Huybrechts S.J. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2009; 284: 10504-10513Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In this system, an organelle suspension (e.g. a postnuclear supernatant) is incubated with 35S-labeled PEX5 under and the of the protein is The data gathered until now with from interaction M. C. K. D. Van Mol. Cell 2002; Full Text Full Text PDF PubMed Scopus Google and and (17Dodt G. Warren D. Becker E. Rehling P. Gould S.J. J. Biol. Chem. 2001; 276: 41769-41781Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (178) Google Scholar, G. Gould S.J. J. Cell Biol. PubMed Scopus Google Scholar, V. Subramani S. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google the pathway C.P. Carvalho A.F. Pinto M.P. T. Sá-Miranda C. Azevedo J.E. Cell Mol. Sci. 2009; PubMed Scopus Google for a recent cytosolic PEX5 synthesized peroxisomal matrix proteins in the cytosol. The PEX5·cargo protein then at the peroxisomal docking/translocation machinery a protein comprising and the proteins and C. Oliveira M.E. A.M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (83) Google Scholar, B. K. H. Rehling P. A. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This interaction in the ATP-independent cargo of PEX5 into the an step M.E. A.M. Pinto R.A. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Carvalho A.F. A.M. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The PEX5 is then monoubiquitinated at a conserved cysteine C. Berg M. Distel B. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, A.F. Pinto M.P. C.P. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). This is a mandatory modification for the next step of the pathway, the ATP-dependent dislocation of monoubiquitinated PEX5 by the receptor a protein and two of the protein with and a peroxisomal protein (21Miyata N. Fujiki Y. Mol. Cell. Biol. 2005; 25: 10822-10832Crossref PubMed Scopus (175) Google Scholar, A.F. Pinto M.P. C.P. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, H.W. S. K. Nat. Cell Biol. 2005; 7: PubMed Scopus Google Scholar). Finally, ubiquitin is removed from the cytosolic conjugate by a of and C.P. Carvalho A.F. Pinto M.P. Huybrechts S.J. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2009; 284: 10504-10513Abstract Full Text Full Text PDF PubMed Scopus (71) Google yielding free PEX5 A protein then at the mechanism of protein translocation across the peroxisomal membrane, the few data the cargo proteins in this the where the cargo protein is from the cytosolic of the peroxisomal into the and from the into the peroxisomal on the that PEX5 at the 2 the majority of its into the peroxisomal matrix A.M. Guimaraes C.P. Oliveira M.E. Reguenga C. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 226-232Abstract Full Text Full Text PDF PubMed Scopus (82) Google and on the that of PEX5 into the is cargo A.M. C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google we that are across the organelle at the 2 J.E. Costa-Rodrigues J. C.P. Oliveira M.E. C. Cell Biochem. Biophys. PubMed Scopus Google Scholar). the that this is the release of the cargo protein from the there are C.P. Carvalho A.F. Pinto M.P. T. Sá-Miranda C. Azevedo J.E. Cell Mol. Sci. 2009; PubMed Scopus Google Scholar, C. Berg M. Distel B. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). A in vitro import system where a 35S-labeled cargo protein is incubated with an organelle suspension of a to this type of mechanistic a few many S. S. Osumi T. T. Fujiki Y. J. Biochem. 1994; PubMed Scopus Google Scholar, T. Lazarow P.B. J. Cell Biol. PubMed Scopus Google Scholar, Y. Lazarow P.B. J. Biol. Chem. Full Text PDF PubMed Google a in the peroxisomal are this P.A. Gould S.J. Subramani S. Mol. Cell. Biol. PubMed Scopus Google in its is its a the and In this we describe a that this in vitro system. that a postnuclear supernatant is with a robust of a 35S-labeled PTS2-containing protein be into The data obtained to the step of protein translocation across the peroxisomal into the PEX5 cycling The large isoform of PEX5 (PEX5L) J. Carvalho A.F. Fransen M. E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google a protein comprising the of C.P. Carvalho A.F. Pinto M.P. S. H. B. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google the A.F. C.P. Pinto M.P. Costa-Rodrigues J. Fransen M. Sá-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2007; 1773: PubMed Scopus Google and the protein A.F. Pinto M.P. C.P. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google obtained the of a protein comprising the of the small isoform of PEX5 the A.F. C.P. Pinto M.P. Costa-Rodrigues J. Fransen M. Sá-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2007; 1773: PubMed Scopus Google used the in a with the and The with and and into the of This protein then in the and using synthesized C.P. Carvalho A.F. Pinto M.P. Huybrechts S.J. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2009; 284: 10504-10513Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The encoding from by using the and to the sequence A. Y. T. J.M. Res. 16: PubMed Scopus Google Scholar). A encoding the sequence of by a obtained by using the and into the and the of proteins synthesized using the in the of the of the The of 35S-labeled proteins obtained with this not to the of are in 1 2 A.M. Guimaraes C.P. Oliveira M.E. Reguenga C. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 226-232Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In a import of protein and 1 of a 35S-labeled for at in of import 2 and 2 the of 35S-labeled a PEX5 proteins and for and ubiquitin to The from a 1 in in and in used at and In the at not import of of cytosolic both the 35S-labeled protein and the with incubated with of cytosolic protein to C.P. Carvalho A.F. Pinto M.P. S. H. B. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google in the of of in of import of at for the of the the of the by to by for an at in import incubated for at in the of of this suspension to of import and ubiquitin in the for and at of the and the incubated for an of import on for using of the with for 2 on the organelle to with and by at The then to and to and the proteins by of the in vitro import in this at of in vitro 35S-labeled into the peroxisomal matrix occurs from in vitro import the of and in for by in a and into two on whereas the other to to from proteins of and to and a The to an to the 35S-labeled protein and with the and and and of to the the of protein in the a of the import and of the the import to with and by step M.P. C.P. Oliveira M.E. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of in 1 and mammalian and A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The and Van E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (126) Google Scholar, M. T. C. Van Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). The to number number and number and on using and The of and their in PTS1-containing proteins PTS1 is not cleaved upon import (1Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (288) Google have the of a robust in vitro import system. The from the that be it a postnuclear supernatant a organelle contain PTS1 proteins that have from the organelle T. Lazarow P.B. J. Cell Biol. PubMed Scopus Google Scholar, Fujiki Y. H. Lazarow P.B. J. Cell Biol. PubMed Scopus Google Scholar). The of proteins in in vitro import few of in from the data in A.M. C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google that an in vitro synthesized 35S-labeled PTS1-containing protein of PEX5 A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google A to this is to import with PEX5. with this the in vitro import for a PTS1 is the of the protein to the the of PTS1 protein complexes now a at the S. T. A. C. P. M. C. and J. E. PTS2 proteins are PTS1 proteins M. N. S. N. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. M. C.P. M. C. H. T. J. Azevedo J.E. B. Mol. Cell 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the PTS2 is proteolytically removed upon import (1Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (288) Google Scholar, 7Kurochkin I.V. Mizuno Y. Konagaya A. Sakaki Y. Schönbach C. Okazaki Y. EMBO J. 2007; 26: 835-845Crossref PubMed Scopus (83) Google Scholar). This one to in vitro import, that a 35S-labeled PTS2-containing protein not from proteins in the interaction with PEX7. cargo protein complexes are to the by and at one of the the with protein complexes at the to this of peroxisomal A to this from the that of not to bind PTS1-containing are in targeting PTS2 proteins to the in (9Braverman N. Dodt G. Gould S.J. Valle D. Hum. Mol. Genet. 1998; 7: 1195-1205Crossref PubMed Scopus (158) Google Scholar, 17Dodt G. Warren D. Becker E. Rehling P. Gould S.J. J. Biol. Chem. 2001; 276: 41769-41781Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). the of a protein with to an in vitro import with a PTS2 protein not to an in the of PTS1 protein that proteins the to have the capacity to the in a cargo process A.M. C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, A.F. C.P. Pinto M.P. Costa-Rodrigues J. Fransen M. Sá-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2007; 1773: PubMed Scopus Google Scholar). the that the obtained with proteins is their in vitro import of a 35S-labeled protein with an organelle suspension under the of the a large of a is to the to 35S-labeled Finally, of the the are and by and 2 the of an using 35S-labeled the PTS2 the system is used in its proteins the of 35S-labeled protein that be in organelle and that of the protein the of under of the import with in a small of 35S-labeled protein with the half of the protein on that 35S-labeled the the of this process is of the data not A obtained a of a protein comprising of and to bind PTS1 proteins in the import The of on import is on the domain it of 35S-labeled protein obtained in import with the the obtained with The the capacity of to bind PTS1 proteins Jr., G.J. Geisbrecht B.V. Gould S.J. Berg J.M. Nat. Biol. 2000; 7: PubMed Scopus Google its PEX7 (9Braverman N. Dodt G. Gould S.J. Valle D. Hum. Mol. Genet. 1998; 7: 1195-1205Crossref PubMed Scopus (158) Google Scholar). import of is and and and the PTS2 present in the in a 35S-labeled protein to not a to in vitro in the of the protein the 35S-labeled protein is a import a and The then and of by and in an peroxisomal a in M.P. C.P. Oliveira M.E. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). is in 2 and of the and the other at and of A.M. Guimaraes C.P. Oliveira M.E. Reguenga C. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 226-232Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, M.P. C.P. Oliveira M.E. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). this is the by 35S-labeled and the 35S-labeled protein into this in vitro import we then not translocation of across the organelle cytosolic valuable on the of this an to this to the translocation step of the cargo protein to a of the PEX5 cycling pathway The to the of a process is to the at which it occurs in the of with that obtained in the of in the of (e.g. of the system used that be into this type of of the in is by PEX5. In the of in the of PEX5 is not from this machinery, and so of import be obtained are used from the of the M.E. A.M. Pinto R.A. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google not this incubated in import for at the of the it with and the 35S-labeled the the species with the in the of in the of be from the and the import capacity of this a large of the ubiquitin in import is used by the on peroxisomal PEX5 at the are not for the ATP-dependent receptor C.P. Carvalho A.F. Pinto M.P. S. H. B. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). in the of the at which the of in be the in all the species not be Finally, a protein is synthesized in an in vitro system, there is that the to a a of that protein is by ATP-dependent M. M. A. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (46) Google Scholar). this not addressed in the 35S-labeled protein used in the with a cytosolic the protein in the of for at so that from ATP-dependent of the cargo interaction be The of this then the import used to not translocation of across the peroxisomal of cytosolic In the the import of obtained with in with ubiquitin and in whereas in the of ubiquitin the of protein obtained in the of is the one obtained in the of 2 and is in and under where dislocation of is a vast of on the of protein a In the we the import of obtained with in with and an that A. M. F. S. M. P.B. Res. PubMed Scopus Google Scholar). in the of 35S-labeled protein is not by the of in the and The in that the of used in from of to an ATP-dependent C.P. Carvalho A.F. Pinto M.P. Huybrechts S.J. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2009; 284: 10504-10513Abstract Full Text Full Text PDF PubMed Scopus (71) Google Interestingly, the of processing in are all that at the N of the protein the matrix of the in a process that dislocation of the receptor from the ubiquitin the of the receptor the data in suggest that the is for the of a major of 35S-labeled In this from in vitro import in the of ubiquitin of the PEX5 cycling pathway ubiquitin is is ubiquitin is by in a and to to proteins from The in an of from the whereas a in the a with of the protein in the and a for in vitro that this of the protein its the matrix of the Interestingly, the majority of not in the this species a with protein (e.g. the peroxisomal S. K. H. H. J. Biochem. 2008; PubMed Scopus Google an species (e.g. a to be The of in an for a suggest that translocation of 35S-labeled across the peroxisomal membrane, by its its processing into the and its release into the peroxisomal matrix, occurs this provides for the the that the PEX5-mediated of a cargo protein into the occurs at the 2 the the that release of the cargo protein into the peroxisomal matrix occurs of the receptor C. Berg M. Distel B. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google the ATP-dependent dislocation of the receptor from the C.P. Carvalho A.F. Pinto M.P. T. Sá-Miranda C. Azevedo J.E. Cell Mol. Sci. 2009; PubMed Scopus Google to the cargo release then release of the cargo protein from the the peroxisomal is to small P. Biochem. J. PubMed Scopus Google Scholar, A. M. 2009; PubMed Scopus (116) Google in with this the import of is to not there is a that the of the peroxisomal matrix is from the one in the due to a Biochim. Biophys. Acta. 2006; 1763: PubMed Scopus (57) Google Scholar). the mechanism for the cargo release step an (e.g. of the in D. M. der J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). and a peroxisomal not to the this are this N. K. S. Fujiki Y. Biochim. Biophys. Acta. 2009; PubMed Scopus Google that translocation of proteins across the peroxisomal of The that from in vitro import where both the postnuclear and the 35S-labeled proteins from of the with a the this type of is to in protein import it is to that in the of a of proteins in of (e.g. and in N. K. S. Fujiki Y. Biochim. Biophys. Acta. 2009; PubMed Scopus Google it the is is not Here we describe a to protein translocation across the mammalian peroxisomal using an in vitro import system. In its proteins this system been used to mechanistic aspects of the PEX5-mediated import pathway using a protein PEX5 The of PEX5 and the of PTS1-containing cargo proteins in the organelle the of the PEX5-centered in vitro system. are the it been so to a robust in vitro import system. import with PEX5 this at the of be by the import with a PTS2-containing protein and one of the used in this it be to a robust in vitro system.