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

Marc Fransen,Chantal Brees,Karen Ghys,Daniel Ladant,Leen Amery,Guy P Mannaerts,Paul P Van Veldhoven

doi:10.1074/mcp.m100025-mcp200

Abstract

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 Pex11pbeta; and (iii) the C(3)HC(4) 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.

Highlights

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis
Building a Peroxin Interaction Matrix reported recently that in S. cerevisiae Pex11p plays a primary role in the medium-chain fatty acid oxidation pathway and suggested the alternative view that this pathway regulates the level of a signaling molecule that modulates peroxisome number and size
Defining the physical interactions between peroxins is an essential step toward the understanding of how these proteins function and how proteins are translocated through the peroxisomal membrane

Summary

EXPERIMENTAL PROCEDURES

Strains and Cell Lines—The E. coli strain Top10FЈ (Invitrogen) was used for all DNA manipulations, as well as for the expression of recombinant proteins. Yeast two-hybrid and protein three-hybrid assays were performed using the S. cerevisiae strain SFY526 (CLONTECH). Two-hybrid and Protein Three-hybrid Analyses—The cotransformation of plasmids in competent SFY526 yeast cells and the colony lift ␤-galactosidase filter assay were performed as described by the manufacturer (CLONTECH). The liquid culture ␤-galactosidase assay with o-nitrophenyl-␤-D-galactopyranoside as substrate was performed as described by the manufacturer (CLONTECH). For this assay the yeast cells were grown for 72 h in minimal dropout medium without leucine and tryptophan (y-2HS) or in minimal dropout medium without leucine and tryptophan in the absence or presence of 1 mM methionine (y-3HS). Bacterial two-hybrid assays were performed as described [25] using the E. coli strain BTH101 (Hybrigenics). Animal experiments were approved by the local institutional ethics committee

RESULTS

Optical density

DISCUSSION