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Vol. 20, Issue 11, 2731-2743, June 1, 2009

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Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling

Marko Jovi, Fabien Kieken, Naava Naslavsky, Paul L. Sorgen, and Steve Caplan

Department of Biochemistry and Molecular Biology, and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870

Submitted November 7, 2008; Revised February 26, 2009; Accepted April 3, 2009
Monitoring Editor: Jennifer Lippincott-Schwartz

The C-terminal Eps15 homology domain (EHD) 1/receptor-mediated endocytosis-1 protein regulates recycling of proteins and lipids from the recycling compartment to the plasma membrane. Recent studies have provided insight into the mode by which EHD1-associated tubular membranes are generated and the mechanisms by which EHD1 functions. Despite these advances, the physiological function of these striking EHD1-associated tubular membranes remains unknown. Nuclear magnetic resonance spectroscopy demonstrated that the Eps15 homology (EH) domain of EHD1 binds to phosphoinositides, including phosphatidylinositol-4-phosphate. Herein, we identify phosphatidylinositol-4-phosphate as an essential component of EHD1-associated tubules in vivo. Indeed, an EHD1 EH domain mutant (K483E) that associates exclusively with punctate membranes displayed decreased binding to phosphatidylinositol-4-phosphate and other phosphoinositides. Moreover, we provide evidence that although the tubular membranes to which EHD1 associates may be stabilized and/or enhanced by EHD1 expression, these membranes are, at least in part, pre-existing structures. Finally, to underscore the function of EHD1-containing tubules in vivo, we used a small interfering RNA (siRNA)/rescue assay. On transfection, wild-type, tubule-associated, siRNA-resistant EHD1 rescued transferrin and β1 integrin recycling defects observed in EHD1-depleted cells, whereas expression of the EHD1 K483E mutant did not. We propose that phosphatidylinositol-4-phosphate is an essential component of EHD1-associated tubules that also contain phosphatidylinositol-(4,5)-bisphosphate and that these structures are required for efficient recycling to the plasma membrane.

Address correspondence to: Steve Caplan (scaplan{at}unmc.edu) or Paul Sorgen (psorgen{at}unmc.edu)

Abbreviations used: EE, early endosome; EHD, Eps15 homology domain; ERC, endocytic recycling compartment; PtdIns4P, phosphatidylinositol-4-phosphate; PtdIns(4,5)P₂, phosphatidylinositol-(4,5)-bisphosphate; Tf, transferrin.

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