SCAMP1 function in endocytosis

Secretory carrier membrane proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMPs contain multiple N-terminal NPF repeats and four highly conserved transmembrane regions. NPF repeats often interact with EH domain proteins that function in budding of transport vesicles from the plasma membrane or the Golgi complex. We now show that the NPF repeats of SCAMP1 bind to two EH domain proteins, intersectin 1, which is involved in endocytic budding at the plasma membrane, and gamma-synergin, which may mediate the budding of vesicles in the trans-Golgi complex. Expression of SCAMP1 lacking the N-terminal NPF repeats potently inhibited transferrin uptake by endocytosis. Our data suggest that one of the functions of SCAMPs is to participate in endocytosis via a mechanism which may involve the recruitment of clathrin coats to the plasma membrane and the trans-Golgi network.     

AtEHDs, novel Arabidopsis EH-domain-containing proteins involved in endocytosis

SUMMARY: Endocytosis is an essential process by which the eukaryotic cell internalizes exogenous material. Studies in yeast and mammalian cells have revealed that endocytosis is a complex molecular process depending on regulated interactions between a variety of proteins and lipids through specific modules. One such module is the Eps15 homology (EH) domain, a conserved modular protein-interaction domain found in several endocytic proteins. The EH-domain-containing proteins function as regulators of endocytosis through their ability to interact with other proteins involved in this process. Here we describe the isolation and characterization of two Arabidopsis EH-domain-containing proteins (AtEHD1 and AtEHD2). We show that the two proteins are involved in endocytosis in plant systems and demonstrate that the Arabidopsis EHD proteins function similarly to mammalian EHDs. Similarly to hEHD2, over-expression of AtEHD2 has an inhibitory effect on endocytosis. While transgenic plants over-expressing AtEHD1 had no detectable phenotype, downregulation of AtEHD1 caused retardation of entry of endocytosed material into plant cells.     

Regulation of clathrin coat assembly by Eps15 homology domain-mediated interactions during endocytosis

Clathrin-mediated endocytosis involves a coordinated series of molecular events regulated by interactions among a variety of proteins and lipids through specific domains. One such domain is the Eps15 homology (EH) domain, a highly conserved protein-protein interaction domain present in a number of proteins distributed from yeast to mammals. Several lines of evidence suggest that the yeast EH domain-containing proteins Pan1p, End3p, and Ede1p play important roles during endocytosis. Although genetic and cell-biological studies of these proteins suggested a role for the EH domains in clathrin-mediated endocytosis, it was unclear how they regulate clathrin coat assembly. To explore the role of the EH domain in yeast endocytosis, we mutated those of Pan1p, End3p, or Ede1p, respectively, and examined the effects of single, double, or triple mutation on clathrin coat assembly. We found that mutations of the EH domain caused a defect of cargo internalization and a delay of clathrin coat assembly but had no effect on assembly of the actin patch. We also demonstrated functional redundancy among the EH domains of Pan1p, End3p, and Ede1p for endocytosis. Of interest, the dynamics of several endocytic proteins were differentially affected by various EH domain mutations, suggesting functional diversity of each EH domain.     

A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

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, membrane-bound 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 traffic 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 tubule-inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin-independent endocytosis.     

The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.     

Lipid remodelling in neuroendocrine secretion

Neuroendocrine cells secrete hormones and polypeptides through a complex membrane trafficking process that involves the transport of specific organelles, called large dense core secretory granules, from the Golgi apparatus to specialised sites at the plasma membrane where these vesicles are successively exocytosed and recaptured by endocytosis through tightly coupled reactions. The minimal machinery required for exocytosis has been defined as SNARE proteins associated with few accessory proteins. On the other side, clathrin and dynamin constitute major components of some of the most important endocytotic pathways. Although many protein contributors of both exocytosis and endocytosis are now identified, their actual interplay is not well resolved. Furthermore, the necessary tight coupling of exocytosis and compensatory endocytosis to maintain membrane homeostasis in neuroendocrine cells is far from being understood. In this review, we focus on the more recently identified role of lipids in these important processes that are above all membrane remodelling events.     

Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.     

EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.     

Endocytosis: EH domains lend a hand

A number of proteins that have been implicated in endocytosis feature a conserved protein-interaction module known as an EH domain. The three-dimensional structure of an EH domain has recently been solved, and is likely to presage significant advances in understanding molecular mechanisms of endocytosis.     

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15

Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.     

Mapping of Eps15 domains involved in its targeting to clathrin-coated pits

Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15 homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs. Thus, targeting of Eps15 to CCPs likely results from the collaboration between EH domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs. Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry. The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly.     

Is lipid translocation involved during endo- and exocytosis?

Stimulation of the aminophospholipid translocase, responsible for the transport of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the plasma membrane, provokes endocytic-like vesicles in erythrocytes and stimulates endocytosis in K562 cells. In this article arguments are given which support the idea that the active transport of lipids could be the driving force involved in membrane folding during the early step of endocytosis. The model is sustained by experiments on shape changes of pure lipid vesicles triggered by a change in the proportion of inner and outer lipids. It is shown that the formation of microvesicles with a diameter of 100-200 nm caused by the translocation of plasma membrane lipids implies a surface tension in the whole membrane. It is likely that cytoskeleton proteins and inner organelles prevent a real cell from undergoing overall shape changes of the type seen with giant unilamellar vesicles. Another hypothesis put forward in this article is the possible implication of the phospholipid 'scramblase' during exocytosis which could favor the unfolding of microvesicles.     

The EH network

The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals. Even though the domain was discovered just 5 years ago, a great deal has been learned regarding its three-dimensional structure and binding specificities. Moreover, a number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell. Interestingly, many of the EH-containing and EH-binding proteins display characteristics of endocytic "accessory" proteins, suggesting that the principal function of the EH network is to regulate various steps in endocytosis. In addition, recent evidence suggests that the EH network might work as an "integrator" of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation.     

The cell fate determinant numb interacts with EHD/Rme-1 family proteins and has a role in endocytic recycling

The adaptor protein Numb is necessary for the cell fate specification of progenitor cells in the Drosophila nervous system. Numb is evolutionarily conserved and previous studies have provided evidence for a similar functional role during mammalian development. The Numb protein has multiple protein-protein interaction regions including a phosphotyrosine binding (PTB) domain and a carboxy-terminal domain that contains conserved interaction motifs including an EH (Eps15 Homology) domain binding motif and alpha-adaptin binding site. In this study we identify the EHD/Rme-1/Pincher family of endocytic proteins as Numb interacting partners in mammals and Drosophila. The EHD/Rme-1 proteins function in recycling of plasma membrane receptors internalized by both clathrin-mediated endocytosis and a clathrin-independent pathway regulated by ADP ribosylation factor 6 (Arf6). Here we report that Numb colocalizes with endogenous EHD4/Pincher and Arf6 and that Arf6 mutants alter Numb subcellular localization. In addition, we present evidence that Numb has a novel function in endosomal recycling and intracellular trafficking of receptors.     

EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.     

AtEHDs in endocytosis

Endocytosis regulates many important and diverse processes in eukaryotic life. EH domain containing proteins function as regulators of endocytosis through protein-protein interactions. Several interactors of mammalian EHDs were identified, including clathrin machinery components. The four human EHD proteins share high homology at the protein level and possess similar domains, but appear to be involved in different stages of intracellular trafficking. EHD1 regulates recycling through the endocytic recycling compartment (ERC). EHD2 has been found to inhibit internalization in mammalians when overexpressed.We have recently investigated the importance of EH domain containing proteins in plant endocytosis. We were able to show that both of the Arabidopsis EHD homologs, termed AtEHD1 and AtEHD2, play important roles in plant endocytosis. Knockdown of AtEHD1 delayed internalization, and overexpression of AtEHD2 inhibited endocytosis. Thus, the function of plant EHDs is highly homologous to that of mammalian EHDs.Key words: endocytosis, endosome, EH domain, EHD1, EHD2, recycling     

Mechanisms of EHD/RME-1 protein function in endocytic transport

The evolutionarily conserved Eps15 homology domain (EHD)/receptor-mediated endocytosis (RME)-1 family of C-terminal EH domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these adenosine triphosphatases function on endosomal membranes in mammals and Caenorhabditis elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large guanosine triphosphatases.     

Endosomes Derived from Clathrin-Independent Endocytosis Serve as Precursors for Endothelial Lumen Formation

Clathrin-independent endocytosis (CIE) is a form of bulk plasma membrane (PM) endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs) utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen.     

I-BAR domain proteins: linking actin and plasma membrane dynamics

Dynamic plasma membrane rearrangements occur during many cellular processes including endocytosis, morphogenesis, and migration. Actin polymerization together with proteins that directly deform membranes, such as the BAR superfamily proteins, is essential for generation of membrane invaginations during endocytosis. Importantly, recent studies revealed that direct membrane deformation contributes also to the formation of plasma membrane protrusions such as filopodia and lamellipodia. Inverse BAR (I-BAR) domain proteins bind phosphoinositide-rich membrane with high affinity and generate negative membrane curvature to induce plasma membrane protrusions. I-BAR domain proteins, such as IRSp53, MIM, ABBA, and IRTKS also harbor many protein-protein interaction modules that link them to actin dynamics. Thus, I-BAR domain proteins may connect direct membrane deformation to actin polymerization in cell morphogenesis and migration.     

Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal

After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.