Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer.
Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer.
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DOI：
10.1091/mbc.e11-12-1059
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发表时间：
2012-06
影响因子：
3.3
通讯作者：
Rosen MK
中科院分区：
文献类型：
--
作者：
Jia D;Gomez TS;Billadeau DD;Rosen MK
WASH复合物控制内体上的肌动蛋白动力学，其功能机制尚不清楚。WASH复合物亚基Fam 21具有许多拷贝的新基序，该基序直接与retromer货物选择性复合物相互作用。FAM 21的内体定位需要反转录体和多价重复元件。Wiskott-Aldrich综合征蛋白（WASPs）控制细胞过程中的肌动蛋白动力学，包括细胞运动，受体介导的内吞作用，细菌入侵和囊泡运输。我们证明，WASH，最近确定的WASP家族蛋白，共定位于内体亚结构域与货物选择性复合物（CSC）的retromer，在那里它调节逆行分选内体中的肌动蛋白依赖性的方式。然而，WASH募集到这些富含逆转录酶的内体亚结构域的机制尚不清楚。在这里，我们表明，WASH调节复合物（SHRC）的一个组成部分，FAM 21，其中包含21个拷贝的一个新的L-F-[D/E]3-10-L-F基序，直接与逆转录CSC蛋白VPS 35相互作用。FAM 21的内体定位是VPS 35依赖性的，并且依赖于FAM 21重复元件的多价性。使用下拉分析和等温量热法的组合，我们证明了单个重复可以结合CSC，不同的FAM 21重复之间的结合亲和力不同。高亲和力重复序列可以通过基序内疏水残基的突变转化为低亲和力重复序列。这些体外数据反映了FAM 21在细胞中逆转录酶包被的囊泡上的定位。我们提出，多价性使FAM 21能够感测膜上的retromer的密度，从而使SHRC募集和随后的肌动蛋白聚合与retromer分选结构域组织/成熟协调。
The WASH complex controls actin dynamics on endosomes, and its functional mechanism is poorly defined. The WASH complex subunit Fam21 bears many copies of a novel motif that directly interacts with the retromer cargo-selective complex. Endosomal localization of FAM21 requires both the retromer and multivalency of the repeat elements. Wiskott–Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E]3-10-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.