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

Deirdre C Lawe,Varsha Patki,Robin Heller-Harrison,David Lambright,Silvia Corvera

doi:10.1074/jbc.275.5.3699

Deirdre C Lawe, Varsha Patki + Show 3 more

Open Access

https://doi.org/10.1074/jbc.275.5.3699

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Abstract

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.

Highlights

Endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosome fusion
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 be present for endosomal binding
Wortmannin inhibits endosome fusion in in vitro assays [13, 14]. These effects coincide with an inhibition of endosome antigen 1 (EEA1) binding to early endosomal membranes [3, 15], which accounts for the inhibition of endosome fusion in vitro, and may account for some of the wortmannin-induced phenotypes observed in intact cells

Summary

Introduction

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. Exogenous EEA1 added to an in vitro endosome fusion assay was sufficient to rescue fusion both from a wortmannin block and from a block induced by removal of Rab by the addition of GDP dissociation inhibitor [19] From these studies it has been proposed that EEA1 is a critical factor required for endosome fusion, where it acts as a docking/tethering protein between vesicles to promote soluble N-ethylmaleimide-sensitive fusion attachment receptor (SNARE)-mediated fusion reactions [20, 21]. The half-life of PtdIns3P and the duration of the GTP-bound state of Rab may influence the rate and extent of the endosome fusion reaction In this regard, it is worth noting that introduction of a GFP tag at the COOH terminus of EEA1 results in the gross enlargement of early endosomes in vivo, coinciding with a loss of specificity for PtdIns3P binding [4]

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