Interactions between EHD Proteins and Rab11-FIP2: A Role for EHD3 in Early Endosomal Transport

Abstract

Eps15 homology domain (EHD) 1 enables membrane recycling by controlling the exit of internalized molecules from the endocytic recycling compartment (ERC) en route to the plasma membrane, similar to the role described for Rab11. However, no physical or functional connection between Rab11 and EHD-family proteins has been demonstrated yet, and the mode by which they coordinate their regulatory activity remains unknown. Here, we demonstrate that EHD1 and EHD3 (the closest EHD1 paralog), bind to the Rab11-effector Rab11-FIP2 via EH-NPF interactions. The EHD/Rab11-FIP2 associations are affected by the ability of the EHD proteins to bind nucleotides, and Rab11-FIP2 is recruited to EHD-containing membranes. These results are consistent with a coordinated role for EHD1 and Rab11-FIP2 in regulating exit from the ERC. However, because no function has been attributed to EHD3, the significance of its interaction with Rab11-FIP2 remained unclear. Surprisingly, loss of EHD3 expression prevented the delivery of internalized transferrin and early endosomal proteins to the ERC, an effect differing from that described upon EHD1 knockdown. Moreover, the subcellular localization of Rab11-FIP2 and endogenous Rab11 were altered upon EHD3 knockdown, with both proteins absent from the ERC and retained in the cell periphery. The results presented herein promote a coordinated role for EHD proteins and Rab11-FIP2 in mediating endocytic recycling and provide evidence for the function of EHD3 in early endosome to ERC transport. INTRODUCTIONInternalization of plasma membrane proteins is a critical event required for multiple physiological cellular processes and its regulation is mediated by a complex web of molecular components (Mellman, 1996;Conner and Schmid, 2003;Benmerah, 2004).Proteins at the plasma membrane are either internalized into clathrin-coated vesicles (Conner and Schmid, 2003;Benmerah, 2004), or they can be internalized independently of clathrin (Nichols and Lippincott-Schwartz, 2001;Naslavsky et al., 2004b). In either case, the internalized vesicles deliver their cargo to the endocytic pathway by fusing with early endosomes (Naslavsky et al., 2004b). Although many proteins are transported along the endosomal pathway to late endosomes and lysosomes where they ultimately undergo degradation, some endocytic receptors are returned to the plasma membrane where they continue to exert their physiological effects (Maxfield and McGraw, 2004).Recycling to the plasma membrane can occur either directly from the early endosome in a process that is not well understood (Sheff et al., 1999;Hao and Maxfield, 2000;Sheff et al., 2002;van Dam et al., 2002) or indirectly through a pericentriolar-localized organelle known as the endocytic recycling compartment (ERC) (Gruenberg and Maxfield, 1995;Maxfield and McGraw, 2004). This compartment is a condensed cellular region containing tubular membrane structures that emanate from the microtubule organizing center (Hopkins and Trowbridge, 1983;Yamashiro et al., 1984).Evidence suggests that endocytic rec...

Discovery of epigenetically silenced genes in acute myeloid leukemias

“…28,29 A recent study has demonstrated that RNAi-mediated knockdown of EHD3 in HeLa cells results in the failure of internalized transferrin to localize to the ERC, instead remaining contained in large, peripheral organelles. 30 In the same study, the authors note recent evidence implying that blocking vesicle trafficking to the ERC may, in fact, increase the rate of recycling. This is thought to occur through a 'fast recycling' pathway.…”

Collapsin Response Mediator Protein-2 (Crmp2) Regulates Trafficking by Linking Endocytic Regulatory Proteins to Dynein Motors

“…Since the single Caenorhabditis elegans ortholog (known as RME-1) was originally identified as a regulator of yolk receptor recycling (2), its closest mammalian homolog, EHD1, was found to regulate the recycling of receptors that traverse both the clathrin-dependent (3,4) and the clathrin-independent (5, 6) internalization pathways. Despite similarities to the Ras family of GTP-binding proteins (5,7), EHD proteins bind and hydrolyze ATP (7)(8)(9), a function necessary for their localization to tubular and vesicular membranes (5,7,10).…”

“…Recent studies have determined that the EH domains of the C-terminal EHD proteins have a highly positively charged surface (12) and selectively interact with NPF motifs followed by clusters of acidic residues (13,14). Moreover, EHD proteins coordinate endocytic transport with Rab proteins through their interactions with common effectors that contain such NPF motifs (9,15). More recently, it was demonstrated that EHD1 interacts with the NPF-containing MICAL family protein, MICAL-L1, a Rab8 effector that localizes to EHD1-containing tubules and regulates endocytic transport and recycling (16).…”

Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria

“…Rab GTPases and Rab effectors are tightly linked in all components of the endosomal trafficking system, and have long been known to coordinate vesicular trafficking (65,66). Furthermore, both Rab and Rab effectors are known binding partners of EHD proteins (46,(67)(68)(69). Currently, there are Ͼ60 Rab GTPases with an equal number of Rab effectors.…”