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J. Biol. Chem., Vol. 280, Issue 26, 24404-24411, July 1, 2005

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Pex5p, the Peroxisomal Cycling Receptor, Is a Monomeric Non-globular Protein^*

João Costa-Rodrigues¶, Andreia F. Carvalho¶, Marc Fransen||, Eva Hambruch**, Wolfgang Schliebs**, Clara Sá-Miranda, and Jorge E. Azevedo

From the Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, Instituto de Ciências Biomédicas de Abel Salazar, Largo do Professor Abel Salazar, 2, 4099-003 Porto, Portugal, ^||Katholieke Universiteit Leuven, Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Herestraat 49, B-3000 Leuven, Belgium, and ^**Institut fur Physiologische Chemie, Ruhr-Universitat Bochum, D-44780 Bochum, Germany

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.

Received for publication, February 22, 2005 , and in revised form, April 15, 2005.

^* This work was supported in part by Fundação para a Ciência e Tecnologia and Fundo Europeu de Desenvolvimento Regional funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by Fundação para a Ciência e Tecnologia, Portugal.

To whom correspondence should be addressed. Tel.: 351-226074900; Fax: 351-226099157; E-mail: jazevedo{at}ibmc.up.pt.

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