The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.     

Contribution of the GTPase Domain to the Subcellular Localization of Dynamin in the Nematode Caenorhabditis elegans

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.     

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.     

Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-golgi network of 3T3L1 adipocytes

To identify the targeting domains of syntaxin 6 responsible for its localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes. Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation. Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN. In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6. Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.     

A conserved mechanism of synaptogyrin localization

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-tagged Caenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.     

Lipidic antagonists to SNARE-mediated fusion

SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.     

Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane

Ca(2+)-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca2+ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca(2+)-dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca(2+)-induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca2+ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca(2+)-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.     

C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane

The protein kinase B(β) (Akt2) pathway is known to?mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138?kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes. CDP138 mutants lacking the Ca(2+)-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.     

Investigating the role played by protein-lipid and protein-protein interactions in the membrane association of the p120GAP CaLB domain.

The GTPase activating protein, p120GAP, contains an amino acid sequence motif called the Ca2+-dependent lipid binding domain (CaLB) which mediates a protein-protein interaction between p120GAP and annexin VI and also binds to negatively charged phospholipids. Because membrane association of p120GAP is important for the regulation of p21 Ras activity, we have studied the roles played by Ca2+, phospholipids and annexin VI in the membrane association of p120GAP. Here we demonstrate that a truncated CaLB domain GST fusion protein (GSTGAP618-632), lacking the ability to bind to phospholipids, is able to bind to rat fibroblast membranes in a Ca2+- and concentration-dependent manner. In addition, this fusion protein also binds to annexin VI in an amino acid sequence specific but Ca2+ independent manner. Also, when bound to annexin VI in the presence of Ca2+, this fusion protein has the ability to co-bind to phosphatidylserine vesicles. Thus, annexin VI may simultaneously mediate an interaction with p120GAP and also an interaction with membrane phospholipids. This may in part explain the mechanism by which p120GAP associates with membranes in response to Ca2+ elevation and suggests the potential importance of annexin VI in the regulation of p21 Ras and the role CaLB domains may play in the specific recognition of cellular membranes.     

Functional Compartmentalization of the Plasma Membrane of Neurons by a Unique Acyl Chain Composition of Phospholipids

In neurons, the plasma membrane is functionally separated into several distinct segments. Neurons form these domains by delivering selected components to and by confining them within each segment of the membrane. Although some mechanisms of the delivery are elucidated, that of the confinement is unclear. We show here that 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), a unique molecular species of phospholipids, is concentrated at the protrusion tips of several neuronal culture cells and the presynaptic area of neuronal synapses of the mouse brain. In PC12 cells, NGF-stimulated neuronal differentiation induces a phospholipase A1 activity at the protrusion tips, which co-localizes with the OPPC domain. Inhibition of the phospholipase A1 activity leads to suppression of phospholipid remodeling in the tip membrane and results in disappearance of the OPPC at the tips. In these cells, confinement of dopamine transporter and Gα_o proteins to the tip was also disrupted. These findings link the lateral distribution of the molecular species of phospholipids to the formation of functional segments in the plasma membrane of neurons and to the mechanism of protein confinement at the synapse.     

Polarized dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize odorant receptors to olfactory cilia

Odorant receptors and signaling proteins are localized to sensory cilia on olfactory dendrites. Using a GFP-tagged odorant receptor protein, Caenorhabditis elegans ODR-10, we characterized protein sorting and transport in olfactory neurons in vivo. ODR-10 is transported in rapidly moving dendritic vesicles that shuttle between the cell body and the cilia. Anterograde and retrograde vesicles move at different speeds, suggesting that dendrites have polarized transport mechanisms. Residues immediately after the seventh membrane-spanning domain of ODR-10 are required for localization; these residues are conserved in many G protein-coupled receptors. UNC-101 encodes a mu1 subunit of the AP-1 clathrin adaptor complex. In unc-101 mutants, dendritic vesicles are absent, ODR-10 receptor is evenly distributed over the plasma membrane, and other cilia membrane proteins are also mislocalized, implicating AP-1 in protein sorting to olfactory cilia.     

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.     

Lipid dependence of ABC transporter localization and function

Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and discussed in terms of relevance to ABC transporter function. We will focus on three ABC transporters of the A, B and C subfamily, respectively. Two of these transporters are relevant to multidrug resistance in tumor cells (Pgp/ABCB1 and MRP1/ABCC1), while the third (ABCA1) is extensively studied in relation to the reverse cholesterol pathway and cellular cholesterol homeostasis. We will attempt to derive a generalized model of lipid rafts to which they associate based on the use of various different lipid raft isolation procedures. In the context of lipid rafts, modulation of ABC transporter localization and function by two relevant lipid classes, i.e. sphingolipids and cholesterol, will be discussed.     

The cytosolic domain of Fis1 binds and reversibly clusters lipid vesicles

Every lipid membrane fission event involves the association of two apposing bilayers, mediated by proteins that can promote membrane curvature, fusion and fission. We tested the hypothesis that Fis1, a tail-anchored protein involved in mitochondrial and peroxisomal fission, promotes changes in membrane structure. We found that the cytosolic domain of Fis1 alone binds lipid vesicles, which is enhanced upon protonation and increasing concentrations of anionic phospholipids. Fluorescence and circular dichroism data indicate that the cytosolic domain undergoes a membrane-induced conformational change that buries two tryptophan side chains upon membrane binding. Light scattering and electron microscopy data show that membrane binding promotes lipid vesicle clustering. Remarkably, this vesicle clustering is reversible and vesicles largely retain their original shape and size. This raises the possibility that the Fis1 cytosolic domain might act in membrane fission by promoting a reversible membrane association, a necessary step in membrane fission.     

Regulation of protein kinases by lipids

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.     

The expression of vesicular glutamate transporters defines two classes of excitatory synapse

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.     

Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.