The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding - Critical role of this dual interaction for endosomal localization
The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding - Critical role of this dual interaction for endosomal localization
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DOI：
10.1074/jbc.275.5.3699
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发表时间：
2000-02-04
影响因子：
4.8
通讯作者：
Corvera, S
中科院分区：
文献类型：
--
作者：
Lawe, DC;Patki, V;Corvera, S
早期内小体抗原1(EEA1)是内小体融合所必需的170 kDa多肽。EEA1在体外可与磷脂酰肌醇3-磷酸(PtdIns3P)和Rab5-GTP结合，但这种双重作用在内膜上的功能作用尚不清楚。在这里，我们已经确定了EEA1与这些配体结合所需的结构特征，我们发现FYVE结构域对PtdIns3P和Rab5结合都是关键的，而PtdIns3P结合只需要FYVE结构域，Rab5结合另外还需要一个与FYVE结构域直接相邻的30个氨基酸的区域。将谷胱甘肽S-转移酶融合结构域显微注射到Cos细胞中发现，仅有FYVE结构域不足以定位到细胞膜上；上游需要30个氨基酸的区域才能与Rab5结合，才能与内体结合。发现野生型Rab5的表达增加增加了EEA1的内体结合，减少了其对PtdIns3P的依赖，从而强调了Rab5在EEA1膜结合中的重要性，因此，Rab5的水平对EEA1募集到内体膜是限速的。PtdIns3P可能在调节Rab5EEA1相互作用中发挥作用。
Early endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosome fusion. EEA1 binds to both phosphtidylinositol 3-phosphate (PtdIns3P) and to Rab5-GTP in vitro, but the functional role of this dual interaction at the endosomal membrane is unclear. Here we have determined the structural features in EEA1 required for binding to these ligands, We have found that the FYVE domain is critical for both PtdIns3P and Rab5 binding, Whereas PtdIns3P binding only required the FYVE domain, Rab5 binding additionally required a 30-amino acid region directly adjacent to the FYVE domain. Microinjection of glutathione S-transferase fusion constructs into Cos cells revealed that the FYVE domain alone is insufficient for localization to cellular membranes; the upstream 30-amino acid region required for Rab5 binding must also be present for endosomal binding. The importance of Rab5 in membrane binding of EEA1 is underscored by the finding that the increased expression of wild-type Rab5 increases endosomal binding of EEA1 and decreases its dependence on PtdIns3P, Thus, the levels of Rab5 are rate-limiting for the recruitment of EEA1 to endosome membranes. PtdIns3P may play a role in modulating the Rab5 EEA1 interaction.