Mapping the Cargo Protein Membrane Translocation Step into the PEX5 Cycling Pathway
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.032565
ISSN1083-351X
AutoresInês S. Alencastre, Tony A. Rodrigues, Cláudia P. Grou, Marc Fransen, Clara Sá-Miranda, Jorge E. Azevedo,
Tópico(s)Signaling Pathways in Disease
ResumoNewly 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 different conditions, and the behavior of the radiolabeled protein is monitored. The data gathered until now together with information coming from protein-protein interaction studies (see Ref. 24Fransen M. Brees C. Ghys K. Amery L. Mannaerts G.P. Ladant D. Van Veldhoven P.P. Mol. Cell Proteomics. 2002; 1: 243-252Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar and references cited therein) and cell biology experiments (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, 25Dodt G. Gould S.J. J. Cell Biol. 1996; 135: 1763-1774Crossref PubMed Scopus (269) Google Scholar, 26Dammai V. Subramani S. Cell. 2001; 105: 187-196Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) support the following pathway (Fig. 1) (see Ref. 27Grou C.P. Carvalho A.F. Pinto M.P. Alencastre I.S. Rodrigues T.A. Freitas M.O. Francisco T. Sá-Miranda C. Azevedo J.E. Cell Mol. Life Sci. 2009; 66: 254-262Crossref PubMed Scopus (53) Google Scholar for a recent review). First, cytosolic PEX5 (stage 0) binds newly synthesized peroxisomal matrix proteins in the cytosol. The PEX5·cargo protein complex (stage 1) then docks at the peroxisomal docking/translocation machinery (DTM), a membrane-embedded protein complex comprising PEX13, PEX14, and the RING finger proteins PEX2, PEX10, and PEX12 (28Reguenga C. Oliveira M.E. Gouveia A.M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2001; 276: 29935-29942Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 29Agne B. Meindl N.M. Niederhoff K. Einwächter H. Rehling P. Sickmann A. Meyer H.E. Girzalsky W. Kunau W.H. Mol. Cell. 2003; 11: 635-646Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). This interaction ultimately results in the ATP-independent but cargo protein-dependent insertion of PEX5 into the DTM (stage 2), an essentially irreversible step (30Oliveira M.E. Gouveia A.M. Pinto R.A. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 39483-39488Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 31Gouveia A.M. Guimarães C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 4389-4392Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 32Costa-Rodrigues J. Carvalho A.F. Gouveia A.M. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2004; 279: 46573-46579Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The DTM-embedded PEX5 is then monoubiquitinated at a conserved cysteine residue (stage 3) (33Williams C. van den Berg M. Sprenger R.R. Distel B. J. Biol. Chem. 2007; 282: 22534-22543Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 34Carvalho A.F. Pinto M.P. Grou C.P. Alencastre I.S. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2007; 282: 31267-31272Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This is a mandatory modification for the next step of the pathway, the ATP-dependent dislocation of monoubiquitinated PEX5 (Ub·PEX5) by the receptor export module, a protein complex containing PEX1 and PEX6, two members of the AAA protein family (ATPases associated with diverse cellular activities family), and PEX26, a peroxisomal membrane protein (21Miyata N. Fujiki Y. Mol. Cell. Biol. 2005; 25: 10822-10832Crossref PubMed Scopus (175) Google Scholar, 34Carvalho A.F. Pinto M.P. Grou C.P. Alencastre I.S. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2007; 282: 31267-31272Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 35Platta H.W. Grunau S. Rosenkranz K. Girzalsky W. Erdmann R. Nat. Cell Biol. 2005; 7: 817-822Crossref PubMed Scopus (189) Google Scholar). Finally, ubiquitin is removed from the cytosolic Ub·PEX5 conjugate (stage 4), probably by a combination of enzymatic and non-enzymatic mechanisms (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) yielding free PEX5 (stage 0). A new protein transport cycle then starts. Ironically, although aiming at describing the mechanism of protein translocation across the peroxisomal membrane, the model described above actually contains very few data regarding the cargo proteins themselves. Two obvious events still missing in this model regard the steps where the cargo protein is 1) moved from the cytosolic side of the peroxisomal membrane into the DTM and 2) released from the DTM into the peroxisomal matrix. Based on the observation that PEX5 at the stage 2 level exposes the majority of its mass into the peroxisomal matrix (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) and on the finding that insertion of PEX5 into the DTM is cargo protein-dependent (31Gouveia A.M. Guimarães C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 4389-4392Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), we previously proposed that cargoes are translocated across the organelle membrane at the stage 1-to-stage 2 transition (36Azevedo J.E. Costa-Rodrigues J. Guimarães C.P. Oliveira M.E. Sã-Miranda C. Cell Biochem. Biophys. 2004; 41: 451-468Crossref PubMed Scopus (25) Google Scholar). However, the fact remains that no direct evidence supporting this idea is presently available. Regarding the release of the cargo protein from the DTM, there are virtually no data, only some hypotheses (27Grou C.P. Carvalho A.F. Pinto M.P. Alencastre I.S. Rodrigues T.A. Freitas M.O. Francisco T. Sá-Miranda C. Azevedo J.E. Cell Mol. Life Sci. 2009; 66: 254-262Crossref PubMed Scopus (53) Google Scholar, 33Williams C. van den Berg M. Sprenger R.R. Distel B. J. Biol. Chem. 2007; 282: 22534-22543Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). A classical in vitro import system where a 35S-labeled cargo protein is incubated with an organelle suspension is, of course, a first choice strategy to address this type of mechanistic question. However, besides a few pioneering studies performed many years ago (37Miura S. Miyazawa S. Osumi T. Hashimoto T. Fujiki Y. J. Biochem. 1994; 115: 1064-1068Crossref PubMed Scopus (24) Google Scholar, 38Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (160) Google Scholar, 39Fujiki Y. Lazarow P.B. J. Biol. Chem. 1985; 260: 5603-5609Abstract Full Text PDF PubMed Google Scholar), such a strategy was never very popular among researchers in the peroxisomal field. There are several reasons explaining why this approach was abandoned (see Ref. 40Walton P.A. Gould S.J. Feramisco J.R. Subramani S. Mol. Cell. Biol. 1992; 12: 531-541Crossref PubMed Scopus (73) Google Scholar), but in essence its main problem is its low yield, a strong limitation when drawing the line between specific and unspecific phenomena. In this work, we describe a simple procedure that dramatically improves this cargo-centered in vitro system. We show that when a rat liver postnuclear supernatant is fortified with selected recombinant proteins, a robust amount of a 35S-labeled PTS2-containing protein can be specifically imported into peroxisomes. The data obtained allowed us to map the step of protein translocation across the peroxisomal membrane into the PEX5 cycling pathway. The recombinant large isoform of human PEX5 (PEX5L) (41Costa-Rodrigues J. Carvalho A.F. Fransen M. Hambruch E. Schliebs W. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2005; 280: 24404-24411Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), a protein comprising the first 324 amino acid residues of PEX5L (ΔC1PEX5L) (42Grou C.P. Carvalho A.F. Pinto M.P. Wiese S. Piechura H. Meyer H.E. Warscheid B. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2008; 283: 14190-14197Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), PEX5L containing the missense mutation N526K (PEX5L(N526K); (43Carvalho A.F. Grou C.P. Pinto M.P. Alencastre I.S. Costa-Rodrigues J. Fransen M. Sá-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2007; 1773: 1141-1148Crossref PubMed Scopus (31) Google Scholar)), and the GST·ubiquitin fusion protein (GST·Ub (34Carvalho A.F. Pinto M.P. Grou C.P. Alencastre I.S. Fransen M. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2007; 282: 31267-31272Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar)) were obtained as described previously. For the production of a histidine-tagged recombinant protein comprising the first 287 amino acid residues of the small isoform of human PEX5 (ΔC1PEX5S), the plasmid pGEM4-PEX5S (43Carvalho A.F. Grou C.P. Pinto M.P. Alencastre I.S. Costa-Rodrigues J. Fransen M. Sá-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2007; 1773: 1141-1148Crossref PubMed Scopus (31) Google Scholar) was used as the template in a PCR reaction with the primers 5′-GCGAACTGCATATGGCAATGCGGGAGCTGG-3′ and 5′-GCGCGGATCCTCATTAGTACCCCTTATCATAGGTAGCTG-3′. The resulting PCR product was digested with NdeI and BamHI and cloned into the NdeI/BamHI sites of pET28c vector (Promega). This protein was then expressed in the Escherichia coli strain BL21 and purified using HIS-SelectTM nickel affinity gel (Sigma). 35S-Labeled PEX5L(C11K) was synthesized as described before (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). The cDNA encoding full-length human thiolase precursor (clone IMAGE ID 3860150, from imaGenes) was amplified by PCR using the primers 5′-GCGAAGCTTGCCACCATGCAGAGGCTGCAGGTA-3′ and 5′-GCGCGAATTCTCAGTTCCCAGGGTATTCAA-3′, designed according to the published sequence (44Bout A. Teunissen Y. Hashimoto T. Benne R. Tager J.M. Nucleic Acids Res. 1988; 16: 10369Crossref PubMed Scopus (48) Google Scholar). A cDNA encoding the amino acid sequence of mature thiolase preceded by a methionine (ΔPTS2 thiolase) was obtained by PCR using the primers 5′-GAATACGAAGCTTGCCACCATGCTGAGCGGTGCC-3′ and 5′-GCGCGAATTCTCAGTTCCCAGGGTATTCAA-3′. Both cDNAs were cloned into the HindIII and the EcoRI sites of pGEM4 (Promega). 35S-Labeled proteins were synthesized using the TnT® T7 quick coupled transcription/translation kit (Promega) in the presence of [35S]methionine (specific activity >1000 Ci/mmol; PerkinElmer Life Sciences) following the standard conditions of the manufacturer. The amounts of 35S-labeled proteins obtained with this kit were not determined, but according to the manufacturer, yields of 3–6 ng/μl are common. Rat liver PNS was prepared in SEM buffer (0.25 m sucrose, 20 mm MOPS-KOH, pH 7.4, 1 mm EDTA-NaOH, pH 7.4, 2 μg/ml N-(trans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide (E-64)) as described before (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). In a typical import reaction, 400 μg of rat liver PNS protein and 1 μl of a rabbit reticulocyte lysate containing 35S-labeled prethiolase or ΔPTS2 thiolase were used. Incubation was for 45 min at 37 °C in 100 μl of import buffer (0.25 m sucrose, 50 mm KCl, 20 mm MOPS-KOH, pH 7.4, 3 mm MgCl2, 20 μm methionine, 2 μg/ml E-64, and 2 mm GSH, pH 7.2) containing 3 mm ATP. GSH increases the amounts of 35S-labeled prethiolase acquiring a protease-resistant status. Recombinant PEX5 proteins (2 and 4 ng/μl for ΔC1PEX5 versions and full-length proteins, respectively; ∼55 nm final concentrations), GST·Ub, or bovine ubiquitin (10 μm) were added to some reactions, as indicated. The ionophores valinomycin (added from a 1 mm stock solution in ethanol), Fluka calcium ionophore II (0.5 mm in ethanol), and carbonylcyanide m-chlorophenylhydrazone (1 mm in ethanol) were used at 10, 5, and 10 μm, respectively. In the experiments aiming at determining whether or not import of prethiolase requires hydrolysis of cytosolic ATP, both the 35S-labeled protein and the PNS were pretreated with ATP as follows. Rabbit reticulocyte lysates were incubated with 40 μg of rat liver cytosolic protein prepared according to Grou et al. (42Grou C.P. Carvalho A.F. Pinto M.P. Wiese S. Piechura H. Meyer H.E. Warscheid B. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2008; 283: 14190-14197Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) in the presence or absence of 200 ng of ΔC1PEX5L in 10 μl of import buffer containing 0.3 mm ATP (quantities per μl of lysate) at 37 °C for 10 min. At the end of the incubation, the energetic status of the solution was either reinforced by adding ATP to 3 mm or changed by adding either ATPγS (3 mm final concentration) or apyrase (20 units/ml final concentration). Incubation proceeded for an additional 10 min. PNS at 8 μg/μl in import buffer was incubated for 5 min at 37 °C in the presence of 0.3 mm ATP. Then, 50-μl aliquots of this suspension were added to 40 μl of import buffer containing ATP (6.75 mm) or ATPγS (6.75 mm) or apyrase (45 units/ml) and either bovine ubiquitin or GST·Ub (22.5 μm each), as specified in the legends for Figs. 4 and 5. After 3 min at 37 °C, 10 μl of the energetically matching rabbit reticulocyte lysate solutions were added, and the mixtures were incubated for an additional 45 min. Protease treatment of import reactions was performed on ice for 45 min using 400 μg/ml trypsin (final concentration). After inactivation of the protease with 500 μg/ml phenylmethylsulfonyl fluoride for 2 min on ice, the organelle suspensions were diluted to 500 μl with SEMK (SEM buffer containing 50 mm KCl) and isolated by centrifugation (11,300 × g, 20 min at 4 °C). The samples were then subjected to SDS-PAGE and transferred to nitrocellulose, and the radioactive proteins were detected by autoradiography. All of the in vitro import experiments reported in this work were performed at least five times.FIGURE 5Release of in vitro imported 35S-labeled thiolase into the peroxisomal matrix occurs before stage 3. Trypsin-treated organelles from in vitro import reactions, containing the indicated combinations of bovine Ub, GST·Ub, ATP, ATPγS, or apyrase and performed as described in legend for Fig. 4, were disrupted by sonication in a low ionic strength buffer and divided into two halves. One-half (samples T) was kept on ice, whereas the other was subjected to ultracentrifugation to separate membranes (samples P) from soluble proteins (samples S). Equivalent portions of samples T, P, and S were subjected to SDS-PAGE and blotted onto a nitrocellulose. The membrane was first exposed to an x-ray film to detect the 35S-labeled protein (top panel) and afterward probed with the following antisera: anti-thiolase (Thiol); anti-catalase (CAT), and anti-cytochrome c (Cyt c). p-T and m-T, precursor and mature forms of thiolase, respectively. Numbers to the left indicate the molecular masses of protein standards in kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For the density gradient centrifugation analysis, a 4-fold scale-up of the standard import reaction was used. After trypsin treatment and inactivation of the protease, the complete import mixture was diluted to 1.5 ml with SEM buffer and analyzed by Nycodenz step gradient centrifugation exactly as described before (45Pinto M.P. Grou C.P. Alencastre I.S. Oliveira M.E. Sá-Miranda C. Fransen M. Azevedo J.E. J. Biol. Chem. 2006; 281: 34492-34502Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Sonication of protease treated organelles in SEM buffer containing 1 μg/μl phenylmethylsulfonyl fluoride and 1:500 (v/v) mammalian protease inhibitors mixture (Sigma) and ultracentrifugation was done as described previously (46Gouveia A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: 32444-32451Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The anti-thiolase and anti-PEX13 antibodies were prepared as described (47Antonenkov V.D. Van Veldhoven P.P. Waelkens E. Mannaerts G.P. J. Biol. Chem. 1997; 272: 26023-26031Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 48Fransen M. Wylin T. Brees C. Mannaerts G.P. Van Veldhoven P.P. Mol. Cell. Biol. 2001; 21: 4413-4424Crossref PubMed Scopus (112) Google Scholar). The antibodies directed to catalase (catalogue number RDI-CATALASEabr; Research Diagnostics, Inc.), KDEL (catalogue number ab12223; Abcam), and cytochrome c (catalogue number 556433; BD Pharmingen) were purchased. Rabbit and mouse antibodies were detected on Western blots using alkaline phosphatase-conjugated anti-rabbit and anti-mouse antibodies (Sigma). The intrinsic fragility of peroxisomes and their high content in PTS1-containing proteins (the 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 Scholar)) have hampered the development of a robust PTS1-centered in vitro import system. The main problem stems from the fact that peroxisome suspensions, be it a postnuclear supernatant or a purified organelle fraction, always contain soluble PTS1 proteins that have leaked from the organelles during tissue homogenization, organelle purification, or even simple manipulation (38Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (160) Google Scholar, 49Alexson S.E. Fujiki Y. Shio H. Lazarow P.B. J. Cell Biol. 1985; 101: 294-304Crossref PubMed Scopus (100) Google Scholar). The presence of these proteins in in vitro import reactions (a few hundreds of nanograms in our PNS-based assay; inferred from the data in Ref. 31Gouveia A.M. Guimarães C.P. Oliveira M.E. Sá-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 4389-4392Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) means that an in vitro synthesized 35S-labeled PTS1-containing reporter protein (3–6 ng/μl; see "Experimental Procedures") has little chance of binding endogenous PEX5 (∼30 ng (46Gouveia A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: 32444-32451Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar)). A partial solution to this problem is to supplement import reactions with recombinant PEX5. However, even with this modification, the in vitro import yield for a PTS1 reporter is still modest, probably because the addition of the recombinant protein to the reaction also increases the concentration of PEX5·endogenous PTS1 protein complexes now creating a competition problem at the DTM. 4I. S. Alencastre, T. A. Rodrigues, C. P. Grou, M. Fransen, C. Sá-Miranda, and J. E. Azevedo, unpublished results. Mammalian PTS2 proteins are much less abundant than PTS1 proteins (50Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 51Wiese S. Gronemeyer T. Ofman R. Kunze M. Grou C.P. Almeida J.A. Eisenacher M. Stephan C. Hayen H. Schollenberger L. Korosec T. Waterham H.R. Schliebs W. Erdmann R. Berger J. Meyer H.E. Just W. Azevedo J.E. Wanders R.J. Warscheid B. Mol. Cell Proteomics. 2007; 6: 2045-2057Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 52Islinger M. Lüers G.H. Li K.W. Loos M. Völkl A. J. Biol. Chem. 2007; 282: 23055-23069Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). More importantly, the PTS2 peptide 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 property, besides providing one extra criterion to assess in vitro import, implies that a 35S-labeled PTS2-containing reporter protein will not face much competition from endogenous proteins in the interaction with endogenous PEX7. However, PEX7·PTS2 cargo protein complexes are transported to the peroxisome also by PEX5L, and thus, at least one of the problems stated above, the competition with PEX5·PTS1 protein complexes at the DTM level, also applies to this class of peroxisomal proteins. A solution to this proble
Referência(s)