Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay

被引:58
作者
Fransen, M
Brees, C
Ghys, K
Amery, L
Mannaerts, GP
Ladant, D
Van Veldhoven, PP
机构
[1] Catholic Univ Louvain, Afdeling Farmacol, Dept Mol Celbiol, B-3000 Louvain, Belgium
[2] Inst Pasteur, Unite Biochem Cellulaire, CNRS, URA 2185,Dept Biochim & Genet Mol, F-75724 Paris, France
关键词
D O I
10.1074/mcp.M100025-MCP200
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 CAAX motif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11 pbeta; 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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页码:243 / 252
页数:10
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