Characterization of the interaction between recombinant human peroxin Pex3p and Pex19p - Identification of Trp-104 in Pex3p as a critical residue for the interaction
Characterization of the interaction between recombinant human peroxin Pex3p and Pex19p - Identification of Trp-104 in Pex3p as a critical residue for the interaction
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DOI：
10.1074/jbc.m706139200
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发表时间：
2008-03-07
影响因子：
4.8
通讯作者：
Kato, Hiroaki
中科院分区：
文献类型：
--
作者：
Sato, Yasuhiko;Shibata, Hiroyuki;Kato, Hiroaki
过氧化物酶体生物生成所需的蛋白质被称为过氧化物蛋白。过氧化物酶Pex3p是一种过氧化物酶体膜蛋白（PMP），参与过氧化物酶体膜生物生成。它作为另一种过氧化物Pex19p的对接受体，Pex19p是新合成的pmp的特异性载体蛋白。在这里，我们从亲和性、化学计量学和Pex3p的结合位点等方面确定了Pex3p- pex19p相互作用的物理化学性质和结合方式。人类Pex3p的胞质结构域是过量生产的，使用大肠杆菌表达系统，并通过两层析步骤高度纯化。凝胶过滤色谱分析和固有色氨酸荧光滴定显示，单体Pex3p和单体Pex19p之间形成了一对一的络合物。与Pex19p结合后，Pex3p的色氨酸荧光光谱显示最大发射波长发生了18 nm的大蓝移。这一结果表明，Pex3p的一个或两个色氨酸残基（Trp-104和Trp-224）直接参与了与Pex19p的结合。我们通过拉下实验和表面等离子体共振分析研究了野生型和色氨酸突变体Pex3p的结合活性。结果表明，野生型和W104A、W104F突变体的K-D值分别为3.4 nM、1080 nM和66.2 nM。与突变的亲和差异影响了它们在pex3 ZPG208细胞中的过氧化物酶体恢复活性。这些发现表明，Trp-104的吲哚环直接与Pex19p相互作用，促进Pex19p- pmp复合物的特异性过氧化物酶体易位。
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.