Bottger G, Barnett P, Klein A T, Kragt A, Tabak H F, Distel B
Department of Biochemistry, University of Amsterdam, Academic Medical Center, Meibergdreef 15 1105 AZ Amsterdam, The Netherlands.
Mol Biol Cell. 2000 Nov;11(11):3963-76. doi: 10.1091/mbc.11.11.3963.
A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W(204)XXQF(208), that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Delta cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.
已描述了许多与过氧化物酶体相关的蛋白质，它们参与蛋白质向过氧化物酶体的导入，其中包括过氧化物酶体靶向信号1（PTS1）蛋白Pex5p的受体、含有Src同源3（SH3）结构域的整合膜蛋白Pex13p以及外周膜蛋白Pex14p。在酿酒酵母中，Pex5p和Pex14p都能够通过其SH3结构域与Pex13p结合。Pex14p含有经典的SH3结合基序PXXP，而Pex5p中不存在该序列。Pex13 - SH3的PXXP结合口袋中保守色氨酸的突变消除了与Pex14p的相互作用，但不影响与Pex5p的相互作用，这表明Pex14p是经典的SH3结构域配体，而Pex5p以另一种方式结合SH3结构域。为了确定Pex5p中的SH3结合位点，我们筛选了一个随机诱变的PEX5文库，以寻找与Pex13 - SH3相互作用丧失的突变体。这些突变均位于Pex5p N端一半的一个小区域内。其中一个改变的残基（F208）是序列W(204)XXQF(208)的一部分，该序列在不同物种的Pex5蛋白之间保守。对Trp204进行定点诱变证实了该基序在识别SH3结构域中的重要作用。Pex5p突变体在体内只能部分恢复pex5Delta细胞中PTS1蛋白的导入。体外结合研究表明，在没有Pex14p的情况下，这些Pex5p突变体无法与Pex13 - SH3相互作用，但在有Pex14p存在时恢复了它们的结合能力，这表明形成了由Pex5p、Pex14p和Pex13 - SH3组成
