Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1
Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1
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DOI：
10.1074/jbc.m109239200
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发表时间：
2002-03-08
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;Chawla, A;Corvera, S
DEArly内体抗原1（EEA1）是哺乳动物细胞内体融合所需的170-kDa多肽。EEA1的COOH末端含有与磷脂酰肌醇3-磷酸（PtdIns-3-P）特异性相互作用的FYVE结构域和邻近FYVE结构域的Rab5 GT3结合区。已经假设EEA1与PtdIns-3-P和Rab5两者的双重相互作用提供将EEA1靶向早期内体所需的特异性。为了检验这一假设，我们产生了截短的（氨基酸1277 - 1411）和全长EEA1结构，在COOH末端含有点突变，损害Rab5，但不Ptdlns3-P结合。这些构建体定位于完整细胞中的内体，与它们的野生型对应物一样有效。此外，截短的构建体（野生型和突变型）的过表达损害了内源性EEA1的功能，导致小的未束缚的内体的积累。这些结果表明，与Rab5的关联对于EEA1的初始结合和拴系功能不是必需的。然而，在比较表达全长野生型或突变型EEA1的细胞中的内体时，揭示了Rab5结合的作用。突变型全长EEA1引起内体簇的积累并抑制由Rab5的持续活性形式（Rab5Q79L）引起的内体的扩大。相反，野生型EEA1与Rab5Q79L的表达增强了这种扩大。因此，内体束缚依赖于EEA1与Ptdlns-3-P的相互作用，并且其与Rab5的相互作用似乎调节随后的融合。
DEarly endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both Ptdlns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277-1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not Ptdlns3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1 The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with Ptdlns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.