Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export
Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export
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DOI：
10.1128/mcb.25.24.10822-10832.2005
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发表时间：
2005-12-01
影响因子：
5.3
通讯作者：
Fujiki, Y
中科院分区：
文献类型：
--
作者：
Miyata, N;Fujiki, Y
Peroxisomal基质蛋白翻译后与Peroxisome靶向信号1受体Pex5一起被导入到Peroxisome中。Pex5的较长异构体Pex5L也运输PEX7-pts2蛋白复合体。卸货后，Pex5返回胞浆。为了研究Pex5功能的分子机制，我们构建了一个含有CHO细胞系核后上清组分的无细胞Pex5易位系统。在使用野生型CHO-K1细胞组分的检测中，S-35标记的Pex5被多轮特异性地输入和输出。从大鼠肝脏分离的过氧化体也证明了S-35-Pex5的导入。三磷酸腺苷对S-35-Pex5的进口不是必需的，但对出口是必不可少的。S-35-Pex5既没有导入到来自环过氧化物素缺陷细胞突变株的过氧体残留物中，也没有导入到缺乏Pex5对接位点的Pex14细胞中。相反，S-35-Pex5被导入到PEX1-、PEX6-和PEX26-缺陷细胞突变的过氧体残基中，包括来自过氧化体生物发生障碍患者的细胞，而S-35-Pex5不能从这些细胞中输出，这表明AAA ATPase家族的Pex1和Pex6及其招募者Pex26是Pex5输出所必需的。此外，我们还用天然蓝聚丙烯酰胺凝胶电泳法分析了过氧化体膜上S-35-PEX5结合的复合体。S-35-Pex5位于两个不同的500 kDa和800 kDa的复合体中，它们由不同的过氧化物组分组成，如Pex14和Pex2，这意味着Pex5在亚复合体之间转移。综上所述，结果表明，Pex5很可能进入过氧酶体，改变其相互作用伙伴，然后利用ATP能量退出。
Peroxisomal matrix proteins are posttranslationally imported into peroxisomes with the peroxisome-targeting signal 1 receptor, Pex5. The longer isoform of Pex5, Pex5L, also transports Pex7-PTS2 protein complexes. After unloading the cargoes, Pex5 returns to the cytosol. To address molecular mechanisms underlying Pex5 functions, we constructed a cell-free Pex5 translocation system with a postnuclear supernatant fraction from CHO cell lines. In assays using the wild-type CHO-K1 cell fraction, S-35-labeled Pex5 was specifically imported into and exported from peroxisomes with multiple rounds. S-35-Pex5 import was also evident using peroxisomes isolated from rat liver. ATP was not required for S-35-Pex5 import but was indispensable for export. S-35-Pex5 was imported neither to peroxisome remnants from RING peroxin-deficient cell mutants nor to those from pex14 cells lacking a Pex5-docking site. In contrast, S-35-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, S-35-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export. Moreover, we analyzed the S-35-Pex5-associated complexes on peroxisomal membranes by blue-native polyacrylamide gel electrophoresis. S-35-Pex5 was in two distinct, 500- and 800-kDa complexes comprising different sets of peroxins, such as Pex14 and Pex2, implying that Pex5 transited between the subcomplexes. Together, results indicated that Pex5 most likely enters peroxisomes, changes its interacting partners, and then exits using ATP energy.