An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

Background  

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear.     

Phosphatidylinositol 4-kinase is a component of glucose transporter (GLUT 4)-containing vesicles.

We recently developed a procedure for immunoisolating insulin-responsive membrane vesicles that contain the muscle/fat glucose transporter isoform, GLUT 4, from rat adipocytes. Utilizing this methodology, we are analyzing the components of these vesicles to gain an understanding of how they are regulated by insulin. In this report we identify a phosphatidylinositol (PtdIns) 4-kinase as a constituent of glucose transporter vesicles (GTVs). This kinase has the biochemical and immunological properties of a type II PtdIns 4-kinase as classified by Endeman et al. (Endemann, G., Dunn, S. N., and Cantley, L. C. (1987) Biochemistry 26, 6845-6852). A monoclonal antibody, 4C5G, which specifically inhibits the type II PtdIns 4-kinase, suppresses 80% of the GTV-PtdIns 4-kinase activity. In addition, the GTVs-PtdIns 4-kinase is maximally activated by the nonionic detergent Triton X-100, at a concentration of 0.2% and is inhibited by adenosine with a Ki of approximately 20-30 microM. We find that the GTVs do not contain any PtdIns4P 5-kinase or diacylglycerol kinase activities, whereas these activities were detected in the plasma membrane. An analysis of the subcellular distribution of PtdIns 4-kinase activity in the rat adipocyte shows that there are similar levels of activity in GTVs, plasma membranes, and the high and low density microsomal fractions, whereas the mitochondria- and nuclei-containing fractions have less than 5% of the activity seen in other fractions. Low density microsomes were subfractionated by sucrose density gradient centrifugation and PtdIns 4-kinase activity was found to correlate closely with the distribution of membrane protein, indicating that the activity is equally distributed throughout this heterogenous population of membranes. PtdIns 4-kinase activity measured in GTVs, plasma membranes, and low density microsomes, was not affected by prior treatment of the intact adipocytes with 35 nM insulin. We postulate that while the GTV-PtdIns 4-kinase is not regulated by insulin, it may play a role in defining the fusogenic properties necessary to mediate membrane movement between the GTVs, plasma membranes, and microsomes.     

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.     

Acyl-coenzyme A dehydrogenases are localized on GLUT4-containing vesicles via association with insulin-regulated aminopeptidase in a manner dependent on its dileucine motif

Insulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55-82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55-82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.     

A role for phospholipase D in GLUT4 glucose transporter translocation

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitope-tagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.     

Identification of GPI anchor attachment signals by a Kohonen self-organizing map

MOTIVATION: Anchoring of proteins to the extracytosolic leaflet of membranes via C-terminal attachment of glycosylphosphatidylinositol (GPI) is ubiquitous and essential in eukaryotes. The signal for GPI-anchoring is confined to the C-terminus of the target protein. In order to identify anchoring signals in silico, we have trained neural networks on known GPI-anchored proteins, systematically optimizing input parameters. RESULTS: A Kohonen self-organizing map, GPI-SOM, was developed that predicts GPI-anchored proteins with high accuracy. In combination with SignalP, GPI-SOM was used in genome-wide surveys for GPI-anchored proteins in diverse eukaryotes. Apart from specialized parasites, a general trend towards higher percentages of GPI-anchored proteins in larger proteomes was observed. AVAILABILITY: GPI-SOM is accessible on-line at http://gpi.unibe.ch. The source code (written in C) is available on the same website. SUPPLEMENTARY INFORMATION: Positive training set, performance test sets and lists of predicted GPI-anchored proteins from different eukaryotes in fasta format.     

Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane (review).

The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking out of the storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.     

Phosphorylation-dependent phospholipase D activity of matrix vesicles

A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for the release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.     

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.     

Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion.     

Differential regulation of apoptosis by caspase-mediated cleavage of phospholipase D isozymes

Phospholipase D (PLD) has been implicated in survival and anti-apoptosis, but the molecular mechanism by which it responds to apoptotic stimuli is poorly unknown. Here, we demonstrate that cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site (DDVD₅₄₅S) and PLD2 is cleaved at two or three sites (PTGD₁₃ELD₁₆S and DEVD₂₈T) in the front of N-terminus. Cleavage of PLD was endogenously detected in post-mortem Alzheimer brain together with activated caspase-3, suggesting the physiological relevance. The cleavage of PLD1 but not PLD2 might act as an inactivating process since PLD1 but not PLD2 activity is significantly decreased during apoptosis, suggesting that differential cleavage of PLD isozymes could affect its enzymatic activity. Moreover, caspase-resistant mutant of PLD1 showed more potent anti-apoptotic capacity than that of wild type PLD1, whereas PLD2 maintained anti-apoptotic potency in spite of its cleavage during apoptosis. Moreover, PLD2 showed more potent anti-apoptotic effect than that of PLD1 in overexpression and knockdown experiments, suggesting that difference in anti-apoptotic potency between PLD1 and PLD2 might be due to its intrinsic protein property. Taken together, our results demonstrate that differential cleavage pattern of PLD isozymes by caspase might affect its enzymatic activity and anti-apoptotic function.     

Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles.Abbreviations FITC fluorescein isothiocyanate - IEP Pinner-envelope protein - OE outer-envelope protein - pSSU precursor form of the small subunit of ribulose bisphosphate carboxylaseoxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank P. Å. Albertsson, Lund, Sweden, for introducing one of us (S. E.) to the technique of phase partitioning. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246) and Fonds der Chemischen Industrie.     

A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein- GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI- PLC, cell-associated gp63 could not be detected in immunoblots. Pulse- chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.     

Putative virulence factors are released in association with membrane vesicles from Burkholderia cepacia

Like many other Gram-negative bacteria, Burkholderia cepacia naturally releases membrane vesicles (n-MVs) during normal growth. Through filtration and differential centrifugation, n-MVs from clinical isolates of the IIIa and V genomovars were isolated and their characteristics compared. Electron microscopy revealed that they were spherical, 30-220 nm in diameter, and bilayered. Virulence factors thought to play a role in pathogenicity (e.g., lipase, phospholipase-N, and protease, including a metalloprotease) were found associated with n-MVs, while peptidoglycan zymogram analysis also revealed 26, 28, 36, and 66 kDa peptidoglycan-degrading enzymes. n-MVs were often contaminated with flagella and pili when isolated by traditional methods, and a new strategy using a linear isopycnic sucrose gradient was utilized. For better characterization, this was applied to a representative genomovar IIIa strain (C5424) and showed that n-MVs consisted of a subset of specific outer membrane and periplasmic proteins as well as lipopoly saccharide possessing only a putative minor O-side chain polymer. This finding suggests that certain components are selected by B. cepacia during n-MV formation, and since some are putative virulence factors, this property could help deliver the factors to tissue, thereby aiding infection.     

Cellugyrin is a marker for a distinct population of intracellular Glut4-containing vesicles

Although Glut4 traffic is routinely described as translocation from an "intracellular storage pool" to the plasma membrane, it has been long realized that Glut4 travels through at least two functionally distinct intracellular membrane compartments on the way to and from the cell surface. Biochemical separation and systematic studies of the individual Glut4-containing compartments have been limited by the lack of appropriate reagents. We have prepared a monoclonal antibody against a novel component protein of Glut4 vesicles and have identified this protein as cellugyrin, a ubiquitously expressed homologue of a major synaptic vesicle protein, synaptogyrin. By means of sucrose gradient centrifugation, immunoadsorption, and confocal microscopy, we have shown that virtually all cellugyrin is co-localized with Glut4 in the same vesicles. However, unlike Glut4, cellugyrin is not re-distributed to the plasma membrane in response to insulin stimulation, and at least 40-50% of the total population of Glut4 vesicles do not contain this protein. We suggest that cellugyrin represents a specific marker of a functionally distinct population of Glut4 vesicles that permanently maintains its intracellular localization and is not recruited to the plasma membrane by insulin.     

V-type ATPase is involved in biogenesis of GLUT4 vesicles

Proton pumps participate in several aspects of endocytic protein trafficking. However, their involvement specifically in the GLUT4 pathway has been a matter of great controversy. Here, we report that incubation of 3T3-L1 adipocytes with specific inhibitors of V-type ATPase, concanamycin A and bafilomycin A1, inhibits insulin-regulated glucose transport and results in accumulation of GLUT4 in heavy, rapidly sedimenting intracellular membranes. Correspondingly, the amount of small responsive GLUT4 vesicles in concanamycin A- and bafilomycin A1-treated cells is decreased. We conclude that these drugs block translocation of GLUT4 in adipose cells by inhibiting formation of small insulin-responsive vesicles on donor intracellular membranes. At the same time, proton pump inhibitors do not affect insulin-dependent translocation of preexisting vesicles or GLUT4 sorting in recycling endosomes. On the contrary, wortmannin acutely inhibits insulin-dependent translocation of the preexisting vesicles but has no effect on vesicle formation.     

Identification of a sialoglycopeptide released by self-digestion from human erythrocyte membranes.

Membranes from human O Rhesus-positive erythrocyte 'ghosts' were tested in vitro for their ability to digest their own glycoproteins. 'Ghost' membranes incubated in Tris/HCl buffer, pH 7.4, release a sialoglycopeptide, which contains glucosamine, galactosamine, galactose and mainly polar amino acids. Chemical composition, molecular size and aggregation properties suggest that this glycopeptide may be a fragment of glycophorin.     

Ecto-protein kinase release differs from cleavage by phospholipases of a glycosyl-phosphatidylinositol membrane anchor.

Ecto-protein kinases have been detected as physiological constituents of cells. One feature of ecto-phosvitin/casein kinase (ecto-PK) is its release from the surface in a soluble form when cells are incubated with exogenous substrate protein. This is interesting in view of the fact that some ecto-enzymes are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Such enzymes are known to be released from the surface through cleavage by a phospholipase activity. We therefore investigated whether bacterial phospholipase C (PI-PLC) was able to release ecto-PK from intact HeLa cells. The data show that whereas alkaline phosphatase, known to be GPI-anchored, was solubilized, the ecto-PK was neither released nor affected in its activity. Another effect of treatment of cells with phospholipases was the formation of diacylglycerol or phosphatidic acid which, however, did not occur when cells were incubated with phosvitin, the condition which induces ecto-PK release. These results coherently indicate that cellular phospholipases are not involved in the release mechanism of ecto-PK. Also, the presence of various protease inhibitors did not affect ecto-PK release. Cross-linking of cell-surface proteins by bifunctional agents of the succinimidyl-type suggest a protein-protein interaction responsible for membrane anchoring of the ecto-PK.