Sequential Roles for Phosphatidylinositol 3-Phosphate and Rab5 in Tethering and Fusion of Early Endosomes via Their Interaction with EEA1

Deirdre C Lawe,Anil Chawla,Eric Merithew,John Dumas,Walter Carrington,Kevin Fogarty,Lawrence Lifshitz,Richard Tuft,David Lambright,Silvia Corvera

doi:10.1074/jbc.m109239200

Deirdre C Lawe, Anil Chawla + Show 8 more

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https://doi.org/10.1074/jbc.m109239200

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Abstract

Early 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 PtdIns-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 PtdIns-3-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 PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.

Highlights

Protein trafficking in the secretory and endocytic pathways requires the coordinated participation of distinct protein classes, among which are tethering proteins and Rab GTPases
The results shown here suggest a temporal order of events in which endosome tethering is mediated by the interaction of endosome antigen 1 (EEA1) with PtdIns-3-P and subsequent endosome fusion is dependent on the interaction of EEA1 with Rab5
To dissect the specific structural requirements for the interaction of EEA1 with Rab5, we built upon previous studies demonstrating that, whereas the FYVE domain of EEA1 is necessary and sufficient for its interaction with PtdIns-3-P, the FYVE domain and an additional stretch of 30 amino acids NH2-terminal to the FYVE domain are necessary for Rab5 binding [9]

Summary

Introduction

Protein trafficking in the secretory and endocytic pathways requires the coordinated participation of distinct protein classes, among which are tethering proteins and Rab GTPases. The dual interaction of EEA1 with PtdIns-3-P and Rab has been hypothesized to confer increased specificity and stability to the association of EEA1 with endosomal membranes [3, 4]. To directly test this hypothesis we have identified critical residues required for the interaction of the EEA1 COOH-terminal domain with Rab, and documented the effect of mutation of these residues on EEA1 localization and endosome fusion dynamics. The results shown here suggest a temporal order of events in which endosome tethering is mediated by the interaction of EEA1 with PtdIns-3-P and subsequent endosome fusion is dependent on the interaction of EEA1 with Rab

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