Characterization of the Interaction between Recombinant Human Peroxin Pex3p and Pex19p
Characterization of the Interaction between Recombinant Human Peroxin Pex3p and Pex19p
复制标题
重组人过氧化物酶 Pex3p 和 Pex19p 之间相互作用的表征
DOI：
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发表时间：
2008
影响因子：
4.8
通讯作者：
H. Kato
中科院分区：
文献类型：
--
作者：
Yasuhiko Sato;H. Shibata;H. Nakano;Yuji Matsuzono;Y. Kashiwayama;Yuji Kobayashi;Y. Fujiki;T. Imanaka;H. Kato
过氧化物酶体生物合成所需的蛋白质称为过氧化物酶。过氧化物酶Pex 3 p是一种过氧化物酶体膜蛋白（PMP），参与过氧化物酶体膜的生物合成。它作为另一种过氧化物酶Pex 19 p的对接受体，Pex 19 p是新合成的PMPs的特异性载体蛋白。在这里，我们已经确定的物理化学性质和Pex 3 p-Pex 19 p相互作用的结合方式，在亲和力，化学计量，和Pex 3 p的结合位点。人Pex 3 p的胞质结构域过量产生，使用大肠杆菌表达系统，并通过两个层析步骤高度纯化。凝胶过滤色谱分析和内源性色氨酸荧光滴定显示，单体Pex 3 p和单体Pex 19 p之间形成一对一的复合物。Pex 3 p的色氨酸荧光光谱显示出由Pex 19 p的结合的最大发射波长的18 nm的大的蓝移。该结果表明，Pex 3 p的一个或两个色氨酸残基（Trp-104和Trp-224）直接参与与Pex 19 p的结合。我们通过下拉试验和表面等离子体共振分析研究了Pex 3 p野生型和色氨酸突变体的结合活性。结果，野生型和W104 A和W104 F突变体分别显示出3.4 nm、1080 nm和66.2 nm的KD值。突变亲和力的差异影响了它们在pex 3 ZPG 208细胞中的过氧化物酶体恢复活性。这些结果表明，Trp-104的吲哚环直接与Pex 19 p相互作用，以促进Pex 19 p-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 KD 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.
影响因子：
2.9
作者：
GILL, SC;VONHIPPEL, PH
通讯作者：
VONHIPPEL, PH