In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes
In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes
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DOI：
10.1016/j.bbamcr.2009.02.007
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发表时间：
2009-05-01
影响因子：
5.1
通讯作者：
Fujiki, Yukio
中科院分区：
文献类型：
--
作者：
Miyata, Non;Hosoi, Ken-ichiro;Fujiki, Yukio
过氧化物酶体靶向信号类型2（PTS 2）受体Pex 7 p将PTS 2蛋白从胞质溶胶转运至过氧化物酶体。我们在此建立了一个无细胞的Pex 7 p易位系统。在使用各自来自野生型CHO-K1和pex 7 ZPG 207细胞的核后上清液部分的测定中，将S-35标记的Pex 7 p导入过氧化物酶体中。使用大鼠肝脏过氧化物酶体也明显存在S-35-Pex 7 p输入。S-35-Pex 7 p未从PTS 2导入缺陷的pex 5 ZPG 231和pex 2 Z65导入过氧化物酶体残留物。当S-35-Pex 5 pL的输入被过量的重组Pex 5 pS抑制时，S-35-Pex 7 p的输入被同时消除，这表明Pex 5 pL是Pex 7 p的转运蛋白，不像酵母辅伴侣蛋白Pex 18 p。S-35-Pex 7 p和S-35-Pex 5 p以ATP非依赖性方式输入，而PTS 1和PTS 2货物蛋白的输入是ATP依赖性的。因此，Pex 7 p的ATP非依赖性输入暗示需要ATP水解的Pex 5 p输出不是其货物募集至过氧化物酶体的限制步骤。PTS 1蛋白输入确实对N-乙基马来酰亚胺不敏感，而Pex 5 p输出对N-乙基马来酰亚胺敏感。两者合计，货物蛋白易位通过过氧化物酶体膜更可能涉及另一个ATP需要的步骤，除了Pex 5 p的出口。此外，在同时输入到过氧化物酶体中时，在免疫沉淀物中检测到S-35-Pex 5 pL和S-35-Pex 7 p以相互不同的比例存在于每种输入机器过氧化物酶（包括Pex 14 p、Pex 13 p和Pex 2 p）中，因此表明Pex 7 p以及Pex 5 p从过氧化物酶体上的初始对接复合物易位到RING复合物。(C)2009 Elsevier B. V.保留所有权利。
Pex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, S-35-labeled Pex7p was imported into peroxisomes. S-35-Pex7p import was also evident using rat liver peroxisomes. S-35-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of S-35-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, S-35-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. S-35-Pex7p as well as S-35-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, S-35-Pex5pL and S-35-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes. (C) 2009 Elsevier B.V. All rights reserved.