Endofin recruits clathrin to early endosomes via TOM1
Endofin recruits clathrin to early endosomes via TOM1
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DOI：
10.1242/jcs.01628
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发表时间：
2005-02
影响因子：
4
通讯作者：
L. Seet;W. Hong
中科院分区：
文献类型：
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作者：
L. Seet;W. Hong
TOM1及其相关蛋白TOM1-like 1(TOM1-L1)和TOM1-like 2(TOM1-L2)是VHS结构域蛋白家族的一个亚家族。我们最近发现，Endofin，一种与早期内体相关的FYVE结构域蛋白，能够将胞浆TOM1募集到内体膜上。为了揭示内毒素介导的TOM1内体募集的生物学后果，我们确定了网状蛋白重链是TOM1的主要相互作用蛋白。TOM1与笼状蛋白的最佳结合涉及三个位点：残基300-321、321-326和推测的362-366残基(362LEDEF366)处的笼状蛋白结合盒。虽然残基321-326可以独立地作为弱分子筛蛋白结合基序发挥作用，但300-321氨基酸残基的缺失或362Leu和364Asp突变为ALA残基降低了分子筛蛋白与TOM1的结合。缺失氨基酸300-322的片段含有362Leu和364Asp的Ala突变，失去了与clathrin相互作用的能力。值得注意的是，Endofin的过度表达导致了网状蛋白[但不是Dynamin，或者适配器蛋白(AP)复合体，AP1，AP2或AP3]的大量和特异性的募集到Endofin阳性的内体上。虽然SARA与endofin同源，但它不与TOM1的C末端区域相互作用。对endofin和sARA嵌合蛋白的检测表明，endofin的C末端一半负责与TOM1的C末端区域相互作用，并负责TOM1和网状蛋白向内体的募集。Endofin与TOM1的C-末端结构域相互作用的能力与网状蛋白募集之间的相关性表明，Enofin可能通过TOM1来招募网状蛋白。事实上，一种由TOM1与来自endofin的两个FYVE结构域融合的嵌合蛋白具有将cathrin招募到内体结构上的能力。此外，我们还表明，亲和纯化的TOM1抗体可以取消网状蛋白与TOM1 C-末端区域的结合。当微量注射到细胞中时，这种抗体减少了网状蛋白的膜结合。综上所述，这些结果表明，TOM1是膜上网状蛋白募集的重要分子，而Enofin能够利用内吞体内的这种募集。
TOM1 and its related proteins, TOM1-like1 (TOM1-L1) and TOM1-like2 (TOM1-L2), constitute a subfamily of the VHS domain protein family. We have recently shown that endofin, a FYVE domain protein associated with the early endosome, is able to recruit cytosolic TOM1 onto endosomal membranes. To reveal the biological consequence of endofin-mediated endosomal recruitment of TOM1, we have identified the clathrin heavy chain as a major interacting protein for TOM1. Optimal clathrin binding by TOM1 involves three sites: residues 300-321, 321-326 and a putative clathrin-binding box at residues 362-366 (362LEDEF366). Although residues 321-326 could function independently as a weak clathrin-binding motif, deletion of amino acids 300-321 or mutation of 362Leu and 364Asp to Ala residues reduced the binding of clathrin to TOM1. A fragment lacking amino acids 300-322 and containing 362Leu and 364Asp to Ala mutations lost the ability to interact with clathrin. Remarkably, overexpression of endofin led to a massive and specific recruitment of clathrin [but not dynamin, or the adaptor protein (AP) complexes, AP1, AP2 or AP3] onto endofin-positive endosomes. Although SARA is homologous to endofin, it did not interact with the C-terminal region of TOM1. Examination of chimeric proteins of endofin and SARA suggests that the C-terminal half of endofin is responsible for interaction with the C-terminal region of TOM1 and for recruitment of TOM1 and clathrin to endosomes. The correlation between the ability of endofin to interact with the C-terminal domain of TOM1 and clathrin recruitment suggests that endofin may recruit clathrin via TOM1. Indeed, a chimeric protein consisting of TOM1 fused to two FYVE domains derived from endofin has the ability to recruit clathrin onto endosomal structures. Moreover, we show that affinity-purified TOM1 antibody can abolish binding of clathrin to the C-terminal region of TOM1. Upon microinjection into cells, this antibody reduced the membrane association of clathrin. These results, taken together, suggest that TOM1 is an important molecule for membrane recruitment of clathrin, and that endofin is able to exploit this recruitment at the endosome.