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Molecular & Cellular Proteomics 1:243-252, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Analysis of Mammalian Peroxin Interactions Using a Non-transcription-based Bacterial Two-hybrid Assay^*

Marc Fransen^,, Chantal Brees, Karen Ghys, Leen Amery^,¶, Guy P. Mannaerts, Daniel Ladant^|| and Paul P. Van Veldhoven^,**

Departement Moleculaire Celbiologie, Afdeling Farmacologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49 (O/N), B-3000 Leuven, Belgium
^|| Département de Biochimie et Génétique Moleculaire, Institut Pasteur, Unité de Biochemie Cellulaire (CNRS URA 2185), 28 rue du Dr. Roux, 75724 Paris CEDEX 15, France

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pß; and (iii) the C₃HC₄ RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions.

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