AWP1/ZFAND6 Functions in Pex5 Export by Interacting with Cys-Monoubiquitinated Pex5 and Pex6 AAA ATPase
AWP1/ZFAND6 Functions in Pex5 Export by Interacting with Cys-Monoubiquitinated Pex5 and Pex6 AAA ATPase
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DOI：
10.1111/j.1600-0854.2011.01298.x
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发表时间：
2012-01-01
期刊：
影响因子：
4.5
通讯作者：
Fujiki, Yukio
中科院分区：
文献类型：
--
作者：
Miyata, Non;Okumoto, Kanji;Fujiki, Yukio
在过氧化物酶体（一种亚细胞器）的生物发生过程中，过氧化物酶体靶向信号1 （PTS1）受体Pex5作为含有PTS1的过氧化物酶体基质蛋白的穿梭受体。然而，受体在过氧化物酶体和细胞质之间穿梭的确切机制仍然难以捉摸，尽管已经确定了许多过氧化物酶参与这一过程。本文采用生化分离和体外Pex5输出实验相结合的方法分离出一个新的因子，并鉴定为AWP1/ZFAND6，一个泛素结合NF-？B调制器。在体外Pex5输出实验中，重组AWP1刺激Pex5输出，抗AWP1抗体干扰Pex5输出。AWP1与Pex6 AAA atp酶相互作用，但不与Pex1Pex6复合物相互作用。AWP1 A20锌指结构域介导了AWP1与半胱氨酸泛素化形式的Pex5的优先结合，而不是与未修饰的Pex5的优先结合。RNA干扰抑制AWP1对pts1蛋白向过氧化物酶体的输入有显著影响。此外，在AWP1敲除的细胞中，Pex5的稳定性下降，类似于Pex1、Pex6和Pex26缺陷患者的成纤维细胞，这些缺陷都是Pex5输出所必需的。综上所述，这些结果确定AWP1是Pex6的一个新的辅助因子，参与过氧化物酶体生物发生过程中Pex5输出的调控。
During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-?B modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.