Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay.
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.
Fransen
Marc
M
Departement Moleculaire Celbiologie, Afdeling Farmacologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49 (O/N), B-3000 Leuven, Belgium. marc.fransen@med.kuleuven.ac.be
Brees
Chantal
C
Ghys
Karen
K
Amery
Leen
L
Mannaerts
Guy P
GP
Ladant
Daniel
D
Van Veldhoven
Paul P
PP
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D008322:Mammals; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D020675:Peroxisomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D008565:Membrane Proteins; D020543:Proteome