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• W2008453524 abstract "In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed. In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed. Peroxisomal matrix proteins are synthesized by cytosolic ribosomes and posttranslationally imported into the organelle. Specific targeting of these proteins to the peroxisome is promoted by one of two receptors, Pex5p or Pex7p (reviewed in Refs. 1Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (82) Google Scholar, 2Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar, 3Sparkes I.A. Baker A. Mol. Membr. Biol. 2002; 19: 171-185Crossref PubMed Scopus (34) Google Scholar, 4Weller S. Gould S.J. Valle D. Annu. Rev. Genomics Hum. Genet. 2003; 4: 165-211Crossref PubMed Scopus (162) Google Scholar). By far the vast majority of these proteins use Pex5p as their import receptor. Proteins belonging to this family possess a tripeptide with the sequence SKL (or a variant) at their C termini, the so-called peroxisomal targeting signal type 1 (PTS1) 1The abbreviations used are: PTS, peroxisomal targeting sequence; GST, glutathione S-transferase; TPR, tetratricopeptide repeat; ATPγS, adenosine 5′-O-(3-thiotriphosphate); SH3, Src homology domain 3; DTT, dithiothreitol. (5Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (889) Google Scholar, 6Lametschwandtner 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 (169) Google Scholar, 7Miura S. Kasuya-Arai I. Mori H. Miyazawa S. Osumi T. Hashimoto T. Fujiki Y. J. Biol. Chem. 1992; 267: 14405-14411Abstract Full Text PDF PubMed Google Scholar). The Pex5p domain involved in this interaction is now well characterized. It comprises six TPR domains present in the C-terminal half of the protein (6Lametschwandtner 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 (169) Google Scholar, 8Brocard C. Kragler F. Simon M.M. Schuster T. Hartig A. Biochem. Biophys. Res. Commun. 1994; 204: 1016-1022Crossref PubMed Scopus (128) Google Scholar, 9Dodt 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 (387) Google Scholar, 10Fransen 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 (163) Google Scholar, 11Gatto Jr., G.J. Geisbrecht B.V. Gould S.J. Berg J.M. Nat. Struct. Biol. 2000; 7: 1091-1095Crossref PubMed Scopus (304) Google Scholar, 12Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 13Terlecky S.R. Nuttley W.M. McCollum D. Sock E. Subramani S. EMBO J. 1995; 14: 3627-3634Crossref PubMed Scopus (155) Google Scholar). PTS1-containing proteins interact with Pex5p while still in the cytosol (reviewed in Refs. 1Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (82) Google Scholar, 2Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar, 3Sparkes I.A. Baker A. Mol. Membr. Biol. 2002; 19: 171-185Crossref PubMed Scopus (34) Google Scholar, 4Weller S. Gould S.J. Valle D. Annu. Rev. Genomics Hum. Genet. 2003; 4: 165-211Crossref PubMed Scopus (162) Google Scholar). The recognition of this cytosolic Pex5p-cargo protein complex by the peroxisomal docking/translocation machinery is the next step in the protein transport pathway mediated by Pex5p. Pex13p and Pex14p as well as the RING finger peroxins Pex2p, Pex10p, and Pex12p are components of this docking/translocation machinery (14Agne B. Meindl N.M. Niederhoff K. Einwachter 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 (198) Google Scholar, 15Albertini M. Girzalsky W. Veenhuis M. Kunau W.H. Eur. J. Cell Biol. 2001; 80: 257-270Crossref PubMed Scopus (52) Google Scholar, 16Reguenga C. Oliveira M.E. Gouveia A.M. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2001; 276: 29935-29942Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The third step in this pathway has been the subject of much controversy. According to some researchers, Pex5p is translocated completely across the peroxisomal membrane together with the cargo it transports (17Szilard R.K. Titorenko V.I. Veenhuis M. Rachubinski R.A. J. Cell Biol. 1995; 131: 1453-1469Crossref PubMed Scopus (97) Google Scholar, 18Dammai V. Subramani S. Cell. 2001; 105: 187-196Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19van der Klei I.J. Hilbrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The other perspective is that the Pex5p protein is inserted into the peroxisomal membrane without crossing it completely. In this model, the cargo protein-binding domain of Pex5p reaches the lumen of the organelle where it releases the cargo protein (reviewed in Refs. 20Azevedo J.E. Costa-Rodrigues J. Guimaraes C.P. Oliveira M.E. Sa-Miranda C. Cell Biochem. Biophys. 2004; 41: 451-468Crossref PubMed Scopus (25) Google Scholar and 21Gould S.J. Collins C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 382-389Crossref PubMed Scopus (93) Google Scholar). Regardless of the true mechanism by which matrix proteins are translocated across the peroxisomal membrane, it is consensually accepted that Pex5p returns to the cytosol to catalyze further rounds of transportation. This last step (the recycling event) is probably the only one requiring energy in the form of ATP hydrolysis (22Oliveira M.E. Gouveia A.M. Pinto R.A. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 39483-39488Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The data supporting this general mechanism, the so-called cycling receptor model, are now numerous (18Dammai V. Subramani S. Cell. 2001; 105: 187-196Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 23Gouveia 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 (81) Google Scholar, 24Dodt G. Gould S.J. J. Cell Biol. 1996; 135: 1763-1774Crossref PubMed Scopus (265) Google Scholar). However, it is evident that our knowledge on many of the “details” of this pathway is still quite poor, leaving room for controversies between researchers in the field. These controversies are not restricted to the translocation step as already stated above. Another point of discussion regards the nature of the Pex5p molecule(s) that interact(s) with the docking/translocation machinery. In principle, formation of a heterodimeric complex involving a Pex5p molecule on one side and one cargo protein on the other should suffice to ensure specific targeting of proteins to the peroxisomal compartment. However, this simple idea has been challenged by a more complex model in which Pex5p is seen as a homotetramer and thus capable of binding up to four PTS1-containing cargo proteins (21Gould S.J. Collins C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 382-389Crossref PubMed Scopus (93) Google Scholar). Because many peroxisomal matrix proteins are oligomers, it was proposed that multivalent interactions would be established between Pex5p and the proteins en route to the peroxisomal matrix, leading to the generation of huge protein complexes. Presumably, these large protein complexes would be formed at the surface of the organelle, resulting in an increase in local concentration of the cargo proteins that would facilitate their translocation across the peroxisomal membrane. More recently, a refined version of this “pre-implex model” has been proposed (25Wang D. Visser N.V. Veenhuis M. van der Klei I.J. J. Biol. Chem. 2003; 278: 43340-43345Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). It was suggested that in Hansenula polymorpha cargo proteins are actually translocated across the peroxisomal membrane by tetrameric Pex5p. After releasing the cargo proteins in the matrix of the peroxisome, Pex5p would monomerize and return to the cytosol. Interestingly, a model in which Pex5p also oscillates between a tetrameric and a monomeric/dimeric form has been described recently in Leishmania donovani (26Madrid K.P. De Crescenzo G. Wang S. Jardim A. Mol. Cell. Biol. 2004; 24: 7331-7344Crossref PubMed Scopus (26) Google Scholar). In this case, however, it was proposed that L. donovani Pex5p mono- or dimerizes upon binding PTS1-containing proteins and tetramerizes when leaving the peroxisomal compartment. In this work, we have studied the properties of human Pex5p when bound to a PTS1-containing peptide/protein. Data suggesting the existence of PTS1-induced conformational alterations on human Pex5p were obtained when in vitro synthesized and recombinant Pex5p were subjected to partial proteolysis. To determine whether binding of a PTS1-containing peptide/protein modulates in some way the assumed oligomeric state of human Pex5p, we have characterized the hydrodynamic properties of this peroxin. In agreement with previous data, human Pex5p was found to behave as a 270-kDa globular protein upon size exclusion chromatography (27Harper C.C. Berg J.M. Gould S.J. J. Biol. Chem. 2003; 278: 7897-7901Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 28Schliebs W. Saidowsky J. Agianian B. Dodt G. Herberg F.W. Kunau W.H. J. Biol. Chem. 1999; 274: 5666-5673Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Strikingly, however, this species represents monomeric Pex5p and not homotetramers, as assumed previously. The abnormal behavior of Pex5p on size exclusion chromatography is just the result of a high frictional ratio, a property that seems to derive from its N-terminal half. The implications of these findings for the mechanism of protein translocation across the peroxisomal membrane are discussed. Synthesis of 35S-labeled Pex5p Proteins—The cDNAs encoding full-length human Pex5p (the large isoform; Ref. 29Braverman N. Dodt G. Gould S.J. Valle D. Hum. Mol. Genet. 1998; 7: 1195-1205Crossref PubMed Scopus (156) Google Scholar) or the C-terminal-truncated version comprising amino acid residues 1–324 of Pex5p (ΔC1-Pex5p) preceded by the T7 RNA polymerase promotor were obtained as described previously (30Gouveia A.M. Guimaraes C.P. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 4389-4392Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Oliveira M.E. Reguenga C. Gouveia A.M. Guimaraes C.P. Schliebs W. Kunau W.H. Silva M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (41) Google Scholar). To obtain a cDNA encoding a N-terminal-truncated version of Pex5p lacking the first 204 amino acid residues (ΔN204-Pex5p), the plasmid pGEM-4-Pex5 (31Oliveira M.E. Reguenga C. Gouveia A.M. Guimaraes C.P. Schliebs W. Kunau W.H. Silva M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (41) Google Scholar) was subjected to PCR using the primers 5′-gcggtcgacatggtggatgaccccaaattggcta-3′ and 5′-gcggtcgactcactggggcaggccaaacata-3′. The amplified fragment was cloned into pGEM®-T Easy Vector according to the manufacturer's instructions (Promega). The recombinant plasmid was digested with SalI, the insert was cloned into the SalI site of pGEM-4 (Promega), and the resulting plasmid was linearized by cutting it with NheI. These Pex5p-encoding DNAs were subjected to in vitro transcription using T7 RNA polymerase (Roche Applied Science). 35S-labeled proteins were synthesized using the translation kit Retic Lysate IVT™ (Ambion) in the presence of Redivue™ l-[35S]methionine (specific activity >1000 Ci/mmol) following the manufacturer's instructions. Recombinant Proteins—For expression of the fusion protein His6-PEX5p, the plasmid pGEM-4-Pex5 (31Oliveira M.E. Reguenga C. Gouveia A.M. Guimaraes C.P. Schliebs W. Kunau W.H. Silva M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (41) Google Scholar) was used as template in a PCR reaction using the forward primer 5′-ccggcatgcgcaatgcgggagctggtgg-3′ and the reverse primer 5′-gcggtcgactcactggggcaggccaaacat-3′. The amplified DNA fragment was cloned into pGEM®-T Easy Vector according to the manufacturer's instructions (Promega). The recombinant plasmid was digested with SphI and SalI, and the insert was cloned into pQE-30 (Qiagen), resulting in the pQE-Pex5 plasmid. Expression was performed in the M15 strain of Escherichia coli. 100-ml cultures were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 2 h at 37 °C. Pelleted cells were cooled on ice and lysed by sonication in 1.5 ml of 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% (w/v) Triton X-100, 1 mm EDTA-NaOH, pH 8.0, 1 mm DTT, 0.1 mg/ml phenylmethylsulfonyl fluoride, and 1:500 (v/v) mammalian protease inhibitor mixture (Sigma). Cell debris were removed by centrifugation (15 min at 10,000 × g), and the clarified supernatant was incubated with 100 μl (bed volume) of HIS-Select™ nickel affinity gel (Sigma) for 2 h at 4 °C. The gel was washed three times with 1.5 ml of 50 mm sodium phosphate, pH 8.0, 150 mm NaCl, and the His6-PEX5p was eluted by washing the beads three times with 600 μl of 50 mm sodium phosphate, pH 8.0, 150 mm NaCl, 50 mm imidazole. The eluted protein was concentrated to ∼50 μl using a Vivaspin 10,000 MWCO PES concentrator (Vivascience), diluted to 600 μl with 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA-NaOH, pH 8.0, 1 mm DTT, and concentrated again. This procedure was repeated two more times. Aliquots of the His6-PEX5p recombinant protein were frozen in liquid nitrogen and stored at –70 °C. To test whether the introduction of this purification tag at the N terminus of human Pex5p interferes with its function, the plasmid pQE-Pex5 was subjected to an expression PCR protocol (32Kain K.C. Orlandi P.A. Lanar D.E. BioTechniques. 1991; 10: 366-374PubMed Google Scholar). In the first PCR the upper primer 5′-gggagagccaccatgagaggatcgcatcac-3′ and the lower primer 5′-gcgtaattaagcttggctgcaggtc-3′ were used. In the second PCR the same lower primer was used together with the primer 5′-gaattctaatacgactcactatagggagagccaccatg-3′. The amplified fragment was subjected to in vitro transcription/translation as described above, and the 35S-labeled His6-tagged Pex5p was used in in vitro import experiments exactly as described before (23Gouveia 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 (81) Google Scholar). These experiments revealed that His6-tagged Pex5p is inserted into the peroxisomal membrane originating Stage 2 Pex5p (when ATP was included in the import reactions) or Stage 2 plus Stage 3 Pex5p (when ATPγS was used). Furthermore, addition of a GST fusion protein containing the TPR domains of Pex5p to the import medium blocked the insertion of this His6-tagged protein into the organelle membrane (data not shown), as described previously for the untagged human Pex5p (see Refs. 23Gouveia 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 (81) Google Scholar and 30Gouveia A.M. Guimaraes C.P. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 4389-4392Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These data suggest that the modified N terminus does not inactivate Pex5p. The synthesis and purification of GST-LKS (GST ending with a nonfunctional PTS1-like sequence), GST-SKL (GST containing a PTS1 signal at the C terminus), and the histidine-tagged SH3 domain of human Pex13p (amino acid residues 236–403; Pex13p-SH3) were described before (30Gouveia A.M. Guimaraes C.P. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2003; 278: 4389-4392Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 33Fransen M. Terlecky S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8087-8092Crossref PubMed Scopus (136) Google Scholar). Protease Assay—Protease treatment of Pex5p was performed in buffer A (50 μl of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, and 1 mm DTT). One μl of the reticulocyte lysates containing the 35S-labeled Pex5p proteins or 5 μg of His6-PEX5p recombinant protein were used per reaction. Peptides (see below) and GST-LKS or GST-SKL were added to 2 μm final concentration. Pex13p-SH3 was used at a final concentration of 1.5 μm. After 10 min at 37 °C, the samples were placed on ice and treated for 15 min with 0.5 μg/ml (for radioactive proteins) or 1 μg/ml (for recombinant Pex5p) of proteinase K. The protease was inactivated with phenylmethylsulfonyl fluoride (0.5 mg/ml), and the proteins were precipitated with trichloroacetic acid (10% (w/v)), washed with acetone, and analyzed by SDS-PAGE. Sucrose Gradient Centrifugation—20 μg of recombinant Pex5p or 3 μl of the reticulocyte lysates containing radioactive Pex5p or ΔC1-Pex5p were incubated in 200 μl of buffer A in the presence of 2 μm GST-LKS or GST-SKL for 10 min at 37 °C. Trace amounts (∼2 μg) of bovine IgGs (6.9 s), bovine serum albumin (4.3 s), soybean trypsin inhibitor (2.3 s), and cytochrome c (1.9 s) were added to the samples as internal standards (numbers in parenthesis represent the sedimentation coefficients). These mixtures were then applied onto the top of a discontinuous sucrose gradient (2.0 ml of 4%, 1.8 ml of 8%, 1.7 ml of 12%, 1.5 ml of 16%, 1.2 ml of 20%, 1 ml of 25%, and 1 ml of 30% (w/v) sucrose in buffer A supplemented with 2 μm GST-LKS or GST-SKL. After centrifugation at 38,000 rpm for 20 h at 4 °C in a TST41.14 swing-out rotor (Sorvall), 13 fractions of 0.8 ml were collected from the bottom of the tube and analyzed by SDS-PAGE. The sedimentation coefficients of the Pex5p proteins were estimated by interpolation from the linear plots of the s values of the standard proteins versus their gradient fractionation volume. Size Exclusion Chromatography—Human Pex5p recombinant protein (20 μg of protein) or in vitro synthesized proteins (1 μl of the corresponding reticulocyte lysates) were incubated in 200 μl of buffer A containing 40 μm peptides (see below) or 10 μm GST-SKL or GST-LKS for 10 min at 37 °C. Protein samples were injected into a Superose 12 HR 10/30 column (Amersham Biosciences) running with 50 mm Tris-HCl, pH 7.5, 0.15 m NaCl, 1 mm EDTA-NaOH, pH 7.5, at 0.5 ml/min. The inclusion of 1 mm DTT in this running buffer did not change the elution profile of the Pex5p proteins (see Ref. 28Schliebs W. Saidowsky J. Agianian B. Dodt G. Herberg F.W. Kunau W.H. J. Biol. Chem. 1999; 274: 5666-5673Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; data not shown). The column was calibrated with the following standards (numbers in parenthesis represent the Stokes radii): thyroglobulin (8.5 nm), ferritin (6.1 nm), aldolase (4.8 nm), bovine serum albumin (3.6 nm), and soybean trypsin inhibitor (2.3 nm). Fractions of 500 μl covering the relevant Stokes radius range were always collected, subjected to trichloroacetic acid precipitation, and analyzed by SDS-PAGE. In some experiments with the recombinant protein the A280 profile of the chromatography revealed the existence of one or two extra protein peaks (corresponding to globular proteins with molecular masses of 130 and 60 kDa) in addition to the “270 kDa” species. Analysis of the corresponding SDS gels showed that only the “270 kDa” peak correlated with His-tagged Pex5p; the other peaks were due to the presence of Pex5p proteolytic fragments (data not shown). These Pex5p preparations were discarded. The Stokes radii of the Pex5p proteins were determined as described (34Laurent T.C. Killander J. J. Chromatogr. 1964; 14: 217-230Crossref Google Scholar). Sedimentation Equilibrium Centrifugation—Recombinant Pex5p (200 μg) was subjected to size exclusion chromatography as described above. Fractions containing the “270-kDa” protein peak were pooled and concentrated to 1 mg/ml using a Vivaspin 10,000 MWCO PES concentrator (Vivascience). Sedimentation equilibrium analysis was performed at the National Centre for Macromolecular Hydrodynamics at the University of Nottingham (UK) using a Beckman XL-A analytical ultracentrifuge equipped with scanning absorption optics. The same analysis was performed using a different version of human Pex5p recombinant protein purified as described before (28Schliebs W. Saidowsky J. Agianian B. Dodt G. Herberg F.W. Kunau W.H. J. Biol. Chem. 1999; 274: 5666-5673Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this protein there is a tobacco etch virus protease cleavage site between the N-terminal His tag and the Pex5p sequence. Sedimentation equilibrium analysis of this recombinant protein (0.4 mg/ml in 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 15 mm MgCl2, 1 mm DTT) was performed in a Beckman XL-A analytical ultracentrifuge at European Molecular Biology Laboratory, Heidelberg, Germany. Miscellaneous—Molecular masses of native proteins (M) and frictional ratios (f/fo) were calculated from their Stokes radii and sedimentation coefficients as described in Ref. 35Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar using Equations 1 and 2 M=6πηNas/(1−υρ)(Eq. 1) and f/fo=a/(3υM/4πN)1/3(Eq. 2) where η is the viscosity of the medium, N is the Avogadro's number, a is the Stokes radius, s is the sedimentation coefficient, v is the partial specific volume of the protein, and ρ is the density of the medium. Partial specific volumes for Pex5p proteins and GST-SKL were calculated using the program SEDNTERP v1.08 (www.jphilo.mailway.com/default.htm). Edman degradation of Pex5p proteolytic fragments was performed by HHMI/Keck Biotechnology Resource Laboratory (New Haven, CT). The peptides CRYHLKPLQSKL (Pep-SKL) and CRYHLKPLQLKS (Pep-LKS) were synthesized by Sigma Genosys. The peptide YQSKL was a kind gift from Dr. W. Nastainczyk, University of Saarland, Germany. Controlled digestion of a protein using proteases may reveal important insights on its structure. In many cases, such assays can even be used to characterize ligand-induced conformational alterations on the protein being studied (e.g. Ref. 36Manciu L. Chang X.B. Buyse F. Hou Y.X. Gustot A. Riordan J.R. Ruysschaert J.M. J. Biol. Chem. 2003; 278: 3347-3356Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Naturally, such strategy can also be applied to the identification of putative ligands of a given protein even if the ligands being tested are proteins themselves. In this case, however, positive results may only be obtained if the protein under study is much more sensitive to the protease than the ligands added to the assay, something that can be easily determined empirically. Using this rationale we have developed a simple procedure to identify and characterize protein-protein interactions involving human Pex5p. As shown below, because of its high sensitivity to proteases (see also Ref. 37Gouveia 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 (101) Google Scholar) Pex5p is a suitable protein to use in this kind of assay. The procedure consists of incubating human Pex5p (obtained either from in vitro transcription/translation reactions or from heterologous expression in E. coli) with the test proteins/peptides, followed by limited proteolysis with proteinase K (see “Materials and Methods” for details). Binding of a PTS1-containing Peptide/Protein to Human Pex5p Changes the Accessibility of Proteinase K to a Region of the Pex5p Protein That Precedes Its PTS1-binding Domain—We started our analysis by characterizing the interaction of Pex5p with PTS1-containing proteins/peptides. In vitro synthesized 35S-Pex5p was incubated with either a PTS1-containing peptide (pep-SKL) or with a negative control peptide (pep-LKS; see “Materials and Methods”) and subjected to treatment with a very low concentration (0.5 μg/ml) of proteinase K. After inactivation of the protease the samples were analyzed by SDS-PAGE and autoradiography. As shown in Fig. 1A, lanes 1 and 2, different proteolytic patterns are observed suggesting that Pex5p bound to pep-SKL displays a different susceptibility to proteinase K. The most evident differences regard the appearance of a 40-kDa doublet protein band when pep-SKL is used in these experiments and a 35-kDa protein band that is particularly abundant when the control peptide is used. The 40-kDa doublet band was also observed when a shorter PTS1-containg peptide (YQSKL) was used in these experiments (data not shown). Exactly the same results were obtained when GST-SKL and GST-LKS were substituted for pep-SKL and pep-LKS, respectively (Fig. 1A, lanes 3 and 4). To characterize the Pex5p-derived fragments described above, we repeated the protease assay but this time using chemical amounts of recombinant human Pex5p. The results presented in Fig. 1B demonstrate that recombinant Pex5p displays the behavior of the in vitro synthesized peroxin. N-terminal sequencing of the 40-kDa doublet protein band generated a mixture of two peptide sequences, as expected (for practical reasons, no efforts were made to resolve the two protein bands in order to obtain independent sequences). The sequences were easily ascribed to amino acid residues 291–296 and 295–300 of human Pex5p. Edman degradation of the 35-kDa protein band also resulted in a mixture of two peptide sequences, indicating the presence in this band of two Pex5p fragments that probably also differ at their C termini. One of these fragments starts with the sequence WLSDYD (residues 308–313) and the other with TYDKDG (residues 319–324) of human Pex5p. Taken together these data indicate that binding of a PTS1-containing peptide/protein to Pex5p changes the accessibility of proteinase K to peptide bonds linking amino acid resides 307–308 and 318–319 of human Pex5p. These amino acid residues precede the first TPR domain of human Pex5p (11Gatto Jr., G.J. Geisbrecht B.V. Gould S.J. Berg J.M. Nat. Struct. Biol. 2000; 7: 1091-1095Crossref PubMed Scopus (304) Google Scholar) by 30 and 19 amino acid residues, respectively. Characterization of the Pex5p-Pex13p Interaction—Pex13p is a component of the peroxisomal docking/translocation machinery that contains a C-terminal SH3 domain facing the cytosolic side of the peroxisomal membrane (reviewed in Refs. 1Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (82) Google Scholar, 2Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar, 3Sparkes I.A. Baker A. Mol. Membr. Biol." @default.
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• W2008453524 title "Pex5p, the Peroxisomal Cycling Receptor, Is a Monomeric Non-globular Protein" @default.
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