Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: Conserved Pex5p WXXYF/Y motifs are critical for matrix protein import
Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: Conserved Pex5p WXXYF/Y motifs are critical for matrix protein import
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DOI：
10.1128/mcb.22.6.1639-1655.2002
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发表时间：
2002-03-01
影响因子：
5.3
通讯作者：
Fujiki, Y
中科院分区：
文献类型：
--
作者：
Otera, H;Setoguchi, K;Fujiki, Y
过氧化物酶体靶向信号1型（PTS 1）受体的两种亚型，称为Pex 5 pS和（37个氨基酸更长）Pex 5 pL，在哺乳动物中表达。Pex 5 pL将PTS 1蛋白和Pex 7 p-PTS 2货物复合物转运到过氧化物酶体膜上的初始Pex 5 p对接位点Pex 14 p，而Pex 5 pS仅转运PTS 1货物。在这里，我们报告了负责与过氧化物酶Pex 7 p，Pex 13 p和Pex 14 p相互作用的功能性Pex 5 p结构域。包含氨基酸残基I至243的N-末端半部分，如Pex 5 pL（1-243），与Pex 7 p、Pex 13 p和Pex 14 p结合，并且足以恢复pex 5细胞突变体的受损的PTS 2输入，而C-末端三肽重复基序是PTS 1结合所需的。N端Pex 5 p具有多个Pex 14 p结合位点。丙氨酸扫描分析的高度保守的7个（6个在Pex 5 pS）五肽WXXXF/Y基序驻留在N-末端区域表明，这些基序是必不可少的Pex 5 p与Pex 14 p和Pex 13 p的相互作用。此外，几个WXXXF/Y基序的突变不影响Pex 5 p的PTS输入恢复活性，这意味着Pex 14 p与所有WXXXF/Y位点的结合不是Pex 5 p-货物复合物易位的先决条件。Pex 5 p通过WXXXF/Y基序2 ~ 4与Pex 13 p的N末端部分结合，而不与C末端SH 3区域结合。PTS 1和PTS 2的导入需要Pex 5 p与Pex 14 p的相互作用，而不需要Pex 13 p，而Pex 5 p与Pex 13 p的结合对于具有PTS 1样信号KANL的过氧化氢酶的导入是必需的。Pex 5 p将PTS 1蛋白募集到Pex 14 p，但不募集到Pex 13 p。Pex 14 p和Pex 13 p与装载PTS 1的Pex 5 p形成复合物，但在未装载货物的Pex 5 p存在下解离，这意味着PTS货物在Pex 14 p下游和Pex 13 p上游的步骤从Pex 5 p释放。因此，Pex 14 p和Pex 13 p很可能形成相互和时间上不同的亚复合物，参与过氧化物酶体基质蛋白的输入。
Two isoforms of the peroxisomal targeting signal type 1 (PTS1) receptor, termed Pex5pS and (37-amino-acid-longer) Pex5pL, are expressed in mammals. Pex5pL transports PTS1 proteins and Pex7p-PTS2 cargo complexes to the initial Pex5p-docking site, Pex14p, on peroxisome membranes, while Pex5pS translocates only PTS1 cargoes, Here we report functional Pex5p domains responsible for interaction with peroxins Pex7p, Pex13p, and Pex14p. An N-terminal half, such as Pex5pL(1-243), comprising amino acid residues I to 243, bound to Pex7p, Pex13p, and Pex14p and was sufficient for restoring the impaired PTS2 import of pex5 cell mutants, while the C-terminal tetratricopeptide repeat motifs were required for PTS1 binding. N-terminal Pex5p possessed multiple Pex14p-binding sites. Alanine-scanning analysis of the highly conserved seven (six in Pex5pS) pentapeptide WXXXF/Y motifs residing at the N-terminal region indicated that these motifs were essential for the interaction of Pex5p with Pex14p and Pex13p. Moreover, mutation of several WXXXF/Y motifs did not affect the PTS import-restoring activity of Pex5p, implying that the binding of Pex14p to all of the WXXXF/Y sites was not a prerequisite for the translocation of Pex5p-cargo complexes. Pex5p bound to Pex13p at the N-terminal part, not to the C-terminal SH3 region, via WXXXF/Y motifs 2 to 4. PTS1 and PTS2 import required the interaction of Pex5p with Pex14p but not with Pex13p, while Pex5p binding to Pex13p was essential for import of catalase with PTS1-like signal KANL. Pex5p recruited PTS1 proteins to Pex14p but not to Pex13p. Pex14p and Pex13p formed a complex with PTS1-loaded Pex5p but dissociated in the presence of cargo-unloaded Pex5p, implying that PTS cargoes are released from Pex5p at a step downstream of Pex14p and upstream of Pex13p. Thus, Pex14p and Pex13p very likely form mutually and temporally distinct subcomplexes involved in peroxisomal matrix protein import.