The Import Competence of a Peroxisomal Membrane Protein Is Determined by Pex19p before the Docking Step

doi:10.1074/jbc.M607183200

The Import Competence of a Peroxisomal Membrane Protein Is Determined by Pex19p before the Docking Step^*

^‡Instituto de Biologia Molecular e Celular (IBMC) Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, the ^§Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Largo do Professor Abel Salazar, 2, 4099-003 Porto, Portugal, and the ^¶Katholieke Universiteit Leuven, Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Herestraat 49, B-3000 Leuven, Belgium

3 To whom correspondence should be addressed: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: 351-226074900; Fax: 351-226099157; E-mail: jazevedo{at}ibmc.up.pt.

Abstract

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.

Footnotes

↵4 The abbreviations used are: PMP, peroxisomal membrane protein; GFP, green fluorescent protein; PNS, postnuclear supernatant; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; ATPγS, adenosine 5′-O-(thiotriphosphate); GTPγS, guanosine 5′-O-(thiotriphosphate).
↵5 C. P. Guimarães, A. M. Gouveia, and J. E. Azevedo, unpublished results.
↵6 C. Reguenga and J. E. Azevedo, unpublished results.
↵7 M. P. Pinto, C. P. Grou, I. S. Alencastre, M. E. Oliveira, C. Sá-Miranda, M. Fransen, and J. E. Azevedo, unpublished observations.
↵* The work done in Porto was supported by Fundação para a Ciência e Tecnologia Grant POCTI2010 and Fundo Europeu de Desenvolvimento Regional (FEDER) Funds, Portugal, and by European Union VI Framework program Grant LSHG-CT-2004-512018, Peroxisomes in Health and Disease. The work done in Leuven was supported by the Flemish Government (Geconcerteerde Onderzoeksacties Grant GOA/2004/08) and Fonds voor Wetenschappelijk Onderzoek Vlaanderen (Onderzoeksproject Grant G.0237.04). 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.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1S and 2S.
↵1 Supported by Fundação para a Ciência e Tecnologia.
↵2 Present address: Instituto de Genética Médica Jacinto Magalhães, Praça Pedro Nunes, 4050-466 Porto, Portugal.
- Received July 28, 2006.
- Revision received September 6, 2006.
The American Society for Biochemistry and Molecular Biology, Inc.