Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins.
Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins.
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DOI：
10.1042/bj20100862
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发表时间：
2010-12-15
期刊：
影响因子：
--
通讯作者：
Kuang J
中科院分区：
文献类型：
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作者：
Zhou X;Si J;Corvera J;Gallick GE;Kuang J
衔接蛋白阿利克斯在逆转录病毒出芽期间将逆转录病毒连接到ESCRT机器。阿利克斯的这种功能需要其与ESCRT-III组分CHMP 4在N-末端Bro 1结构域和逆转录病毒Gag蛋白在中间V结构域的相互作用。由于细胞质或重组阿利克斯在非变性条件下不能与CHMP 4或逆转录病毒Gag蛋白相互作用，我们构建了阿利克斯截短和突变以定义阿利克斯与这些伴侣蛋白相互作用被禁止的内在机制。我们的研究结果表明，在Bro 1结构域中的补丁2和TSG 101对接位点的Pro丰富的域之间的分子内相互作用锁定阿利克斯成一个封闭的构象，使阿利克斯无法与CHMP 4和逆转录病毒Gag蛋白相互作用。通过异位表达分子内相互作用位点之一的结合配偶体或通过缺失这些位点之一来缓解阿利克斯的分子内相互作用，促进阿利克斯与这些配偶体蛋白的相互作用并促进阿利克斯与膜的结合。具有组成型开放构象的GFP-ALIX突变体而非野生型蛋白质的异位表达增加了EIAV从HEK 293细胞出芽。这些发现预示，解除阿利克斯的自抑制性分子内相互作用是阿利克斯参与逆转录病毒出芽的关键步骤。
The adaptor protein ALIX links retroviruses to ESCRT machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 docking site in the Pro-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP-ALIX mutant with a constitutively open conformation, but not the wild type protein increases EIAV budding from HEK293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.