Endosomal recruitment of the WASH complex: Active sequences and mutations impairing interaction with the retromer
Endosomal recruitment of the WASH complex: Active sequences and mutations impairing interaction with the retromer
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DOI：
10.1111/boc.201200038
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发表时间：
2013-05-01
影响因子：
2.7
通讯作者：
Gautreau, Alexis
中科院分区：
文献类型：
--
作者：
Helfer, Emmanuele;Harbour, Michael E.;Gautreau, Alexis
Wiskott-Aldrich综合征蛋白和疤痕同源物（WASH）复合物是核内体表面主要的Arp2/3激活因子。分支肌动蛋白网络，由WASH复合体诱导，有助于货物分类和运输中间体的分裂，运往大多数内体路线。一个主要的挑战是了解WASH分子机器是如何被招募到核内体表面的。我们和其他人已经提出后转录内体机制在这一过程中发挥作用。结果在这项工作中，我们使用了一种无偏倚的方法来鉴定WASH复合物的内体受体。我们已经描绘了FAM21亚基的一个短片段，它能够取代内体中的内源性WASH复合物。使用蛋白质组学方法，我们已经确定了逆转录物货物选择复合物（CSC）作为替代内源性WASH复合物的活性FAM21序列的伙伴。FAM21中的一个点突变会消除CSC相互作用，也会损害WASH复合物的位移活性。CSC由VPS35、VPS29和VPS26三个亚基组成。FAM21直接结合后转录CSC的VPS35亚基。此外，我们发现VPS35的一个点突变体阻断了与VPS29的结合，也阻止了与FAM21和WASH复合体的结合，揭示了VPS35 - VPS29相互作用在调节反转录体与WASH复合体的结合中的新作用。这种内源性WASH置换的新方法证实了先前的建议，即逆转录物是内体表面WASH复合物的受体，并确定了介导这种相互作用的关键残基。这两种内体机制（WASH复合体和反转录酶）之间的相互作用可能在内体表面形成平台以有效分拣货物方面发挥关键作用。
Background information The Wiskott-Aldrich syndrome protein and scar homolog (WASH) complex is the major Arp2/3 activator at the surface of endosomes. The branched actin network, that the WASH complex induces, contributes to cargo sorting and scission of transport intermediates destined for most endosomal routes. A major challenge is to understand how the WASH molecular machine is recruited to the surface of endosomes. The retromer endosomal machinery has been proposed by us and others to play a role in this process. Results In this work, we used an unbiased approach to identify the endosomal receptor of the WASH complex. We have delineated a short fragment of the FAM21 subunit that is able to displace the endogenous WASH complex from endosomes. Using a proteomic approach, we have identified the retromer cargo selective complex (CSC) as a partner of the active FAM21 sequence displacing the endogenous WASH complex. A point mutation in FAM21 that abolishes CSC interaction also impairs WASH complex displacement activity. The CSC is composed of three subunits, VPS35, VPS29 and VPS26. FAM21 directly binds the VPS35 subunit of the retromer CSC. Additionally, we show that a point mutant of VPS35 that blocks binding to VPS29 also prevents association with FAM21 and the WASH complex revealing a novel role for the VPS35VPS29 interaction in regulating retromer association with the WASH complex. Conclusions This novel approach of endogenous WASH displacement confirms previous suggestions that the retromer is the receptor of the WASH complex at the surface of endosomes and identify key residues that mediate this interaction. The interaction between these two endosomal machineries, the WASH complex and the retromer, is likely to play a critical role in forming platforms at the surface of endosomes for efficient sorting of cargoes.