The Eps15 homology (EH) domain-containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin-mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin-independent pathway regulated by the small GTP-binding protein ADP-ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membranebound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N-terminal P-loop domain or deletion of the C-terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC-I) molecules that normally traf®c through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC-I recycling. These observations suggest an additional function of EHD1 as a tubuleinducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrinindependent endocytosis. Keywords: Arf6/clathrin-independent/EHD1/MHC class I/recycling IntroductionEndocytic receptors such as the epidermal growth factor (EGF) receptor and the transferrin receptor contain signals within their cytoplasmic domains that mediate their rapid internalization from the plasma membrane (for reviews see Trowbridge et al., 1993;Bonifacino and Dell'Angelica, 1999). Internalization of these receptors is effected by a complex molecular machinery comprising clathrin and various clathrin-associated proteins (for reviews see Kirchhausen, 2000;Brodsky et al., 2001). These proteins assemble on the cytoplasmic face of the membrane to form a supramolecular complex known as a clathrin coat, which recruits the plasma membrane receptors by virtue of interactions with their endocytic signals. Clathrin-coated domains of the plasma membrane undergo invagination and eventually pinch off as clathrincoated vesicles. These vesicles carry the internalized receptors to the early endosomal system, from where some receptors (e.g. the EGF receptor) are targeted to late endosomes and then lysosomes for degradation, while others (e.g. the transferrin receptor) are recycled back to the plasma membrane via a morphologically distinct organelle known as the endosomal recycling compartment (ERC) (for a review see Gruenberg and Max®eld, 1995).Many other plasma membrane proteins lack conventional endocytic signals, but can nonetheless undergo internalization via clathrin-independent pathways (for a review see ). The mechanisms involved in clathrin-independent endocytosis are not well understood. Among the mechanisms that have been invoked for this type of internalization are uptake through non-coated invaginations of the membrane known as caveolae (Kurzchalia and Parton, 1999), endocytosis via lipid rafts , micropinocytosis (Lamaze and Schmid, 1995) and macropinocytosis (Hewlett et al., 1...
Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to pre-ciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle (CV) formation from smaller distal appendage vesicles (DAV). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated CV assembly. Interestingly, only after CV assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step whereby EHD1 and EHD3 reorganize the M-centriole and associated DAV prior to coordinated ciliary membrane and axoneme growth.
Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.
Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently, less was known about the four mammalian dynamin-like C-terminal Eps15 Homology Domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field.
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...
Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.
EHD1 has been implicated in the recycling of internalized proteins to the plasma membrane. However, the mechanism by which EHD1 mediates recycling and its relationship to Rab-family-controlled events has yet to be established. To investigate further the mode of EHD1 action, we sought to identify novel interacting partners. GST-EHD1 was used as bait to isolate a approximately 120-kDa species from bovine and murine brain cytosol, which was identified by mass spectrometry as the divalent Rab4/Rab5 effector Rabenosyn-5. We mapped the sites of interaction to the EH domain of EHD1, and the first two of five NPF motifs of Rabenosyn-5. Immunofluorescence microscopy studies revealed that EHD1 and Rabenosyn-5 partially colocalize to vesicular and tubular structures in vivo. To address the functional roles of EHD1 and Rabenosyn-5, we first demonstrated that RNA interference (RNAi) dramatically reduced the level of expression of each protein, either individually or in combination. Depletion of either EHD1 or Rabenosyn-5 delayed the recycling of transferrin and major histocompatibility complex class I to the plasma membrane. However, whereas depletion of EHD1 caused the accumulation of internalized cargo in a compact juxtanuclear compartment, Rabenosyn-5-RNAi caused its retention within a dispersed peripheral compartment. Simultaneous RNAi depletion of both proteins resulted in a similar phenotype to that observed with Rabenosyn-5-RNAi alone, suggesting that Rabenosyn-5 acts before EHD1 in the regulation of endocytic recycling. Our studies suggest that Rabenosyn-5 and EHD1 act sequentially in the transport of proteins from early endosomes to the endosomal recycling compartment and back to the plasma membrane.
RME-1/EHD1 family proteins are key residents of the recycling endosome required for endosome to plasma membrane transport in C. elegans and mammals. Recent studies suggest parallels of the RME-1/EHD proteins to the Dynamin GTPase superfamily of mechanochemical pinchases that promote membrane fission. Here we show that that endogenous C. elegans AMPH-1, the only C. elegans member of Amphiphysin/BIN1 family of BAR-domain proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1 deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 NPF (D/E) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1/Amphyphysin 2 in EHD1-regulated endocytic recycling. In vitro we find that the purified recombinant AMPH-1/RME-1 complexes produce short, coated, membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, likely through functions required for the production of cargo carriers exiting the recycling endosome for the cell surface.
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