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J. Biol. Chem., Vol. 283, Issue 10, 6136-6144, March 7, 2008

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Characterization of the Interaction between Recombinant Human Peroxin Pex3p and Pex19p

IDENTIFICATION OF TRP-104 IN Pex3p AS A CRITICAL RESIDUE FOR THE INTERACTION^*

Yasuhiko Sato¹, Hiroyuki Shibata, Hiroaki Nakano, Yuji Matsuzono^¶, Yoshinori Kashiwayama^||, Yuji Kobayashi^**, Yukio Fujiki^¶, Tsuneo Imanaka^||, and Hiroaki Kato²

From the Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan, the Department of Cardiac Physiology, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan, the ^¶Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, JST, CREST, Japan, the ^||Department of Biological Chemistry, Graduate School of Medicine & Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan, and the ^**Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Proteins required for peroxisome biogenesis are termed peroxins. The peroxin Pex3p is a peroxisomal membrane protein (PMP), involved in peroxisomal membrane biogenesis. It acts as a docking receptor for another peroxin Pex19p, which is a specific carrier protein for newly synthesized PMPs. Here we have determined the physicochemical properties and binding manners of Pex3p-Pex19p interaction, in terms of the affinity, the stoichiometry, and the binding site in Pex3p. The cytosolic domain of human Pex3p was overproduced, using an Escherichia coli expression system and was highly purified by two chromatography steps. Gel filtration chromatography analyses and intrinsic tryptophan fluorescence titrations revealed that a one-to-one complex is formed between monomeric Pex3p and monomeric Pex19p. The tryptophan fluorescence spectrum of Pex3p showed a large 18-nm blue shift of the maximum emission wavelength by the binding of Pex19p. This result indicates that either one or two tryptophan residues of Pex3p (Trp-104 and Trp-224) are directly involved in binding to Pex19p. We investigated the binding activities of the wild-type and tryptophan mutants of Pex3p by pull-down assays and surface plasmon resonance analyses. As a result, the wild-type and the W104A and W104F mutants showed K_D values of 3.4 nM, 1080 nM, and 66.2 nM, respectively. The affinity differences with mutation affected their peroxisome restoring activities in pex3 ZPG208 cells. These findings suggest that the indole ring of Trp-104 directly interacts with Pex19p to facilitate the specific peroxisomal translocation of the Pex19p-PMP complexes.

Received for publication, July 26, 2007 , and in revised form, December 10, 2007.

^* This work was supported in part by Protein 3000 project and Target Protein Research Program from Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Grants-in-Aid for Scientific Research from Japan Society for Promotion of Science and MEXT, a CREST grant from Science and Technology Agency of Japan, and the Global COE Program from MEXT. 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. S1–S3.

¹ Supported by the 21st Century COE Program "Knowledge Information Infrastructure for Genome Science" from MEXT.

² To whom correspondence should be addressed: Dept. of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4617; Fax: 81-75-753-9272; E-mail: katohiro{at}pharm.kyoto-u.ac.jp.

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