Light Microsomes Research Articles

Proteome analysis of human embryonic stem cells organelles

As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. SignificanceMass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells.

An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.

Self-assembly of Glut4 Storage Vesicles during Differentiation of 3T3-L1 Adipocytes

Glut4 storage vesicles (GSVs) represent translocation-competent vesicular carriers in fat and skeletal muscle cells that deliver Glut4 to the plasma membrane in response to insulin stimulation. GSVs include three major cargo proteins: Glut4, insulin-responsive aminopeptidase (IRAP), and sortilin. Previous work has suggested that the lumenal interaction between Glut4 and sortilin and the cytoplasmic interaction between sortilin and GGA adaptors play an important role in recruitment of Glut4 into the GSVs. However, the mechanism of IRAP targeting to this compartment remains unknown. To address this question, we show that in differentiating adipocytes IRAP enters the GSVs from the "donor" membranes on day 3 of differentiation. Forced expression of sortilin in undifferentiated cells does not recruit IRAP into the vesicles. However, double expression of sortilin and Glut4 reconstitutes functional GSVs that incorporate endogenous IRAP. To explain this process, we show by a yeast two-hybrid system and chemical cross-linking that the lumenal domain of IRAP can interact with the lumenal loop of Glut4. IRAP without the lumenal domain is faithfully targeted to the donor membranes but has significantly lower insulin responsiveness than full-length IRAP. We suggest that lumenal interactions between Glut4 and IRAP play an important role in the assembly of the GSVs.

Plectin 1 links intermediate filaments to costameric sarcolemma through β-synemin, α-dystrobrevin and actin

In skeletal muscles, the sarcolemma is possibly stabilized and protected against contraction-imposed stress by intermediate filaments (IFs) tethered to costameric sarcolemma. Although there is emerging evidence that plectin links IFs to costameres through dystrophin-glycoprotein complexes (DGC), the molecular organization from plectin to costameres still remains unclear. Here, we show that plectin 1, a plectin isoform expressed in skeletal muscle, can interact with beta-synemin, actin and a DGC component, alpha-dystrobrevin, in vitro. Ultrastructurally, beta-synemin molecules appear to be incorporated into costameric dense plaques, where they seem to serve as actin-associated proteins rather than IF proteins. In fact, they can bind actin and alpha-dystrobrevin in vitro. Moreover, in vivo immunoprecipitation analyses demonstrated that beta-synemin- and plectin-immune complexes from lysates of muscle light microsomes contained alpha-dystrobrevin, dystrophin, nonmuscle actin, metavinculin, plectin and beta-synemin. These findings suggest a model in which plectin 1 interacts with DGC and integrin complexes directly, or indirectly through nonmuscle actin and beta-synemin within costameres. The DGC and integrin complexes would cooperate to stabilize and fortify the sarcolemma by linking the basement membrane to IFs through plectin 1, beta-synemin and actin. Besides, the two complexes, together with plectin and IFs, might have their own functions as platforms for distinct signal transduction.

The Interaction of Akt with APPL1 Is Required for Insulin-stimulated Glut4 Translocation

APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1) is an Akt/protein kinase B-binding protein involved in signal transduction and membrane trafficking pathways for various receptors, including receptor tyrosine kinases. Here, we establish a role for APPL1 in insulin signaling in which we demonstrate its interaction with Akt2 by co-immunoprecipitation and pulldown assays. In primary rat adipocytes and skeletal muscle, APPL1 and Akt2 formed a complex that was dissociated upon insulin stimulation in both tissues. To investigate possible APPL1 function in adipocytes, we analyzed Akt phosphorylation, 2-deoxyglucose uptake, and Glut4 translocation by immunofluorescence following APPL1 knockdown by small interfering and short hairpin RNAs. We show that APPL1 knockdown suppressed Akt phosphorylation, glucose uptake, and Glut4 translocation. We also tested the effect in 3T3-L1 adipocytes of expressing full-length APPL1 or an N- or a C-terminal APPL1 construct. Interestingly, expression of full-length APPL1 and its N terminus suppressed insulin-stimulated 2-deoxyglucose uptake and Glut4 translocation to roughly the same extent (40-60%). We confirmed by cellular fractionation that Glut4 translocation was substantially blocked in 3T3-L1 adipocytes transfected with full-length APPL1. By cellular fractionation, APPL1 was localized mainly in the cytosol, and it showed a small degree of re-localization to the light microsomes and nucleus in response to insulin. By immunofluorescence, we also show that APPL1 partially co-localized with Glut4. These data suggest that APPL1 plays an important role in insulin-stimulated Glut4 translocation in muscle and adipose tissues and that its N-terminal portion may be critical for APPL1 function.

The Glucose Transporter 4-regulating Protein TUG Is Essential for Highly Insulin-responsive Glucose Uptake in 3T3-L1 Adipocytes

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.

Optical Manipulation Reveals Strong Attracting Forces at Membrane Contact Sites between Endoplasmic Reticulum and Chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Altered subcellular distribution of IRS-1 and IRS-3 is associated with defective Akt activation and GLUT4 translocation in insulin-resistant old rat adipocytes

Insulin receptor signal transduction depends on the precise intracellular localization of signalling molecules. This study examines the compartmentalization and the insulin-induced translocation and tyrosine phosphorylation of insulin receptor substrates (IRS-1 and IRS-3) in epididymal white adipose tissue from adult and insulin-resistant old rats. We found that insulin induces the translocation of IRS-1 from plasma membrane (PM) and light microsomes (LM) to cytosol, whereas IRS-3 translocates from PM to LM and cytosol upon insulin stimulation. Old rat adipocytes are characterized by higher relative levels of IRS proteins, under basal conditions, in those fractions where they are intended to translocate in response to insulin and exhibit a higher phosphotyrosine content of IRS-1 and -3 in basal conditions and a lower maximal phosphorylation in response to insulin. Furthermore, old rat adipocytes are also characterized by a reduced ability of insulin to stimulate both, Akt/PKB activity and translocation of GLUT4 to the PM. We conclude that the lower stimulation of downstream insulin signalling involved in glucose metabolism in old rat adipocytes may be explained, at least in part, by the altered subcellular distribution of IRS-1 and -3 proteins. In addition, our data suggest that the mechanism of turning on/off insulin receptor-mediated signal is impaired with aging.

A 60-kDa protein abundant in adipocyte caveolae.

To search for caveolar proteins, mice were immunised with rat adipocyte membranes. Hybridoma supernatants were screened for antibodies to proteins on the cytosolic face of caveolae by indirect immunoelectron microscopy of immunogold-labelled adipocyte plasma membrane sheets adsorbed on electron-microscope (EM) grids. One of the hybridoma supernatants (2F11) produced a specific labelling of caveolae which was much more intense than that obtained with caveolin-1 antibodies. In Western blots of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins in crude membrane fractions from different rat tissues, 2F11 labelled a band corresponding to 60 kDa. The intensity of 2F11 labelling was high in adipose tissue and in other tissues varied in parallel to caveolin- labelling. In blots of plasma membrane (PM) and light-microsomal (LM) fractions from a homogenate of adipocytes, prior insulin stimulation of the adipocytes translocated GLUT-4 from the LM to the PM fraction, but was without effect on the distribution of the 60-kDa protein labelled by 2F11. Digestion with endoproteinase lys-C produced the same pattern of immunoreactive fragments of the protein in the vesicular PM and LM fractions, indicating similar membrane topology of the 2F11-reactive, 60-kDa protein in vesicles of PM and LM fractions.

Troglitazone induces GLUT4 translocation in L6 myotubes.

A number of studies have demonstrated that insulin resistance in the skeletal muscle plays a pivotal role in the insulin resistance associated with obesity and type 2 diabetes. A decrease in GLUT4 translocation from the intracellular pool to the plasma membranes in skeletal muscles has been implicated as a possible cause of insulin resistance. Herein, we examined the effects of an insulin-sensitizing drug, troglitazone (TGZ), on glucose uptake and the translocation of GLUT4 in L6 myotubes. The prolonged exposure (24 h) of L6 myotubes to TGZ (10(-5) mol/l) caused a substantial increase in the 2-deoxy-[3H]D-glucose (2-DG) uptake without changing the total amount of the glucose transporters GLUT4, GLUT1, and GLUT3. The TGZ-induced 2-DG uptake was completely abolished by cytochalasin-B (10 micromol/l). The ability of TGZ to translocate GLUT4 from light microsomes to the crude plasma membranes was greater than that of insulin. Both cycloheximide treatment (3.5 x 10(-6) mol/l) and the removal of TGZ by washing reversed the 2-DG uptake to the basal level. Moreover, insulin did not enhance the TGZ-induced 2-DG uptake additively. The TGZ-induced 2-DG uptake was only partially reversed by wortmannin to 80%, and TGZ did not change the expression and the phosphorylation of protein kinase B; the expression of protein kinase C (PKC)-lambda, PKC-beta2, and PKC-zeta; or 5'AMP-activated protein kinase activity. a-Tocopherol, which has a molecular structure similar to that of TGZ, did not increase 2-DG uptake. We conclude that the glucose transport in L6 myotubes exposed to TGZ for 24 h is the result of an increased translocation of GLUT4. The present results imply that the effects of troglitazone on GLUT4 translocation may include a new mechanism for improving glucose transport in skeletal muscle.

GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.

GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins.

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.

Insulin-Dependent Phosphorylation of a 70-kDa Protein in Light Microsomes from Rat Adipocytes

In order to discover possibly novel insulin receptor substrates and/or downstream targets in the insulin signaling pathway, we established a cell-free system for this purpose using purified insulin receptor and subcellular fractions from rat adipocytes as a sourse of cellular substrates. Under these conditions, we have found a 70-kDa protein (pp70) in fat cells that is tyrosine-phosphorylated by the activated insulin receptor. Using sucrose velocity gradient sedimentation we also show that pp70 cofractionate a particulate fraction containing IRS-1 but not with GLUT-4 vesicle-enriched fractions. Our results suggest that pp70 may be an endogenous substrate for the insulin receptor tyrosine kinase.

Dynamics of Protein-tyrosine Phosphatases in Rat Adipocytes

Protein-tyrosine phosphatases (PTPases) play a key role in maintaining the steady-state tyrosine phosphorylation of the insulin receptor (IR) and its substrate proteins such as insulin receptor substrate 1 (IRS-1). However, the PTPase(s) that inactivate IR and IRS-1 under physiological conditions remain unidentified. Here, we analyze the subcellular distribution in rat adipocytes of several PTPases thought to be involved in the counterregulation of insulin signaling. We found that the transmembrane enzymes, protein-tyrosine phosphatase (PTP)-alpha and leukocyte common antigen-related (LAR), were detected predominantly in the plasma membrane and to a lesser extent in the heavy microsomes, a distribution similar to that of insulin receptor. PTP-1B and IRS-1 were present in light microsomes and cytosol, whereas SHPTP2/Syp was exclusively cytosolic. Insulin induced a redistribution of PTP-alpha from the plasma membrane to the heavy microsomes in a parallel fashion with the receptor. The distribution of PTP-1B in the light microsomes from resting adipocytes was similar to that of IRS-1 as determined by sucrose velocity gradient fractionation. Analysis of the catalytic activity of partially purified rat adipocyte PTP-alpha and LAR and recombinant PTP-1B showed that all three PTPases dephosphorylate IR. When a mix of IR/IRS-1 was used as a substrate, PTP-1B was particularly effective in dephosphorylating IRS-1. Considering that IR and IRS-1 can be dephosphorylated in internal membrane compartments from rat adipocytes (Kublaoui, B., Lee, J., and Pilch, P.F. (1995) J. Biol. Chem. 270, 59-65) and that PTP-alpha and PTP-1B are the respective PTPases in these fractions, we conclude that these PTPases are responsible for the counterregulation of insulin signaling there, whereas both LAR and PTP-alpha may act upon cell surface insulin receptors.

Insulin Increases the Association of Akt-2 with Glut4-containing Vesicles

Expression of a constitutively active, membrane-associated Akt-1 (PKB alpha) construct in 3T3L1 adipocytes was shown to induce glucose uptake in the absence of insulin by stimulating Glut4 translocation to the plasma membrane (Kohn, A. D., Summers, S. A., Birnbaum, M. J., and Roth, R. A. (1996) J. Biol. Chem. 271, 31372-31378). However, in rat fat cell the vast majority of Akt-1 is cytosolic and shows no re-distribution to the plasma membrane in response to insulin. On the other hand, little work has been done with other Akt family members such as Akt-2 (PKB beta) or Akt-3 (PKB gamma). In this report, an analysis of the subcellular distribution of Akt-2 in rat adipocytes shows that Akt-2 is present in significant amounts in various membrane compartments, as well as in the cytosol, and the former include the light microsomes where Glut4 is present in the basal state. The distribution of Akt-2 in resting adipocytes was found to substantially overlap with that of Glut4 when light microsomes were subfractionated by a sucrose velocity gradient indicating possible co-localization. We confirmed co-localization of Akt-2 and Glut4 in the basal state by immunopurification of Glut4 vesicles, which exhibited a 5.5-fold increase in Akt-2 in response to insulin relative to the amount of Glut4. These results are consistent with the possibility that Akt-2 may be involved in Glut4 vesicle translocation.

Glut4 Is Targeted to Specific Vesicles in Adipocytes of Transgenic Mice Overexpressing Glut4 Selectively in Adipose Tissue

Adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue (Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4-containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulin-sensitive vesicles as in normal cells.

Regulation of cell surface GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes

The effects of insulin and IGF-I on the cell surface quantities of GLUT1, GLUT3 and GLUT4 glucose transporters in L6 myotubes were determined with the exofacial bis-mannose phololabel (ATB-BMPA). In basal cells, an equal molar quantity of each transporter isoform was found at the cell surface. Insulin stimulated the translocation of all three glucose transporter isoforms to the plasma membrane fraction from the light microsome fraction, resulting in equal molar quantities on the cell surface. IGF-I stimulated a similar translocation of all isoforms, augmented by an increase in surface GLUT3 as assessed by ATB-BMPA.

Cellubrevin Is a Resident Protein of Insulin-sensitive GLUT4 Glucose Transporter Vesicles in 3T3-L1 Adipocytes

Insulin stimulates glucose transport in muscle and fat cells by inducing translocation of GLUT4 glucose transporters from a storage site to the cell surface. The mechanism of this translocation and the identity of the storage site are unknown, but it has been hypothesized that transporters recycle between an insulin-sensitive pool, endosomes, and the cell surface. Upon cell homogenization and fractionation, the storage site migrates with light microsomes (LDM) separate from the plasma membrane fraction (PM). Cellubrevin is a recently identified endosomal protein that may be involved in the reexocytosis of recycling endosomes. Here we describe that cellubrevin is expressed in 3T3-L1 adipocytes and is more abundant in the LDM than in the PM. Cellubrevin was markedly induced during differentiation of 3T3-L1 fibroblasts into adipocytes, in parallel with GLUT4, and the development of insulin regulated traffic. In response to insulin, the cellubrevin content decreased in the LDM and increased in the PM, suggesting translocation akin to that of the GLUT4 glucose transporter. Vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin-II, a protein associated with regulated exocytosis in secretory cells, also redistributed in response to insulin. Both cellubrevin and VAMP-2 were susceptible to cleavage by tetanus toxin. Immunopurified GLUT4-containing vesicles contained cellubrevin and VAMP-2, and immunopurified cellubrevin-containing vesicles contained GLUT4 protein, but undiscernible amounts of VAMP-2. These observations suggest that cellubrevin and VAMP-2 are constituents of the insulin-regulated pathway of membrane traffic. These results are the first demonstration that cellubrevin is present in a regulated intracellular compartment. We hypothesize that cellubrevin and VAMP-2 may be present in different subsets of GLUT4-containing vesicles.

Expression of vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin II and cellubrevin in rat skeletal muscle and in a muscle cell line

Molecular studies have identified a family of synaptic vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins) which have been implicated in synaptic vesicle docking and/or fusion with plasma membrane proteins. Here we demonstrate the expression of two members of this family, VAMP-2/synaptobrevin II and cellubrevin, in skeletal muscle, a tissue with both constitutive and regulated membrane traffic. The 18 kDa VAMP-2 polypeptide was detected in purified membrane fractions from adult skeletal muscle and from L6 myotubes in culture, demonstrating that the presence of this protein in the isolated muscle membrane fractions is not the result of contamination by ancillary tissues such as peripheral nerve. Furthermore, skeletal muscle and the muscle cell line also expressed cellubrevin, a VAMP-2 homologue of 17 kDa; which is much less abundant in brain cells. Both VAMP-2 and cellubrevin were preferentially isolated in membrane fractions rich in plasma membranes, and were less concentrated in light microsomes and other internal membrane fractions of mature muscle or muscle cells in culture. Interestingly, both VAMP-2 and cellubrevin were much more abundant in the differentiated L6 myotubes than in their precursor myoblasts, suggesting that they are required for functions of differentiated muscle cells. The identity of both polypeptides was further confirmed by their susceptibility to proteolysis by Clostridium tetanus toxin. Expression of these products was further established by the presence of mRNA transcripts of VAMP-2 and cellubrevin, but not of VAMP-1, in both skeletal muscle and L6 myotubes. In contrast, other synaptic vesicle and docking/fusion components were undetectable, such as VAMP-1, SNAP25 and syntaxin 1A/1B, as were synaptophysin and synapsin Ia/Ib, proteins which are believed to be involved in sensing the signal for neuronal exocytosis. It is concluded that VAMP-2 and cellubrevin are expressed in skeletal muscle cells and may each participate in specific processes of intracellular membrane traffic.

Allosteric regulation of the Golgi inosine diphosphatase from corn roots

Light microsomes of corn roots, enriched in endoplasmic reticulum and Golgi membranes, have an IDPase activity which is stimulated by Triton X-100 and by cold storage. In the native state, the enzyme activity does not follow Michaelis-Menten kinetics. It hydrolyses IDP with K0.5 of about 900 μM and Vmax of 300–400 nmol Pi/mg protein/min. In the presence of Triton X-100, the enzyme is maximally stimulated and it renders to a Michaelis-Menten behavior with a Km of about 500 μM and a Vmax of 800–1200 nmol Pi/mg protein/min. The maximal effect of the detergent occurs at about 1 mM IDP (270%), being reduced (190%) at high IDP concentrations (>2 mM) which, per se, have a slight stimulatory effect on the enzyme. On the other hand, we observed that ATP (>2 mM) and adenosine inhibit the IDPase. The effects of the nucleotides and of the adenosine are abolished in the presence of Triton X-100, which makes the enzyme fully active. Furthermore, we observed that detergent treatment of the membranes reduces the change in the activation energy which occurs at 10 °C and eliminates cooperative effects, as revealed by the Arrhenius analysis and the Hill analysis, respectively. We also observed that IDPase inhibition by ATP is maximal at low IDP concentrations (1 mM), whereas it decreases at high concentrations of IDP (4 mM), which promote maximal velocities in the native enzyme. Conversely, the inhibitory effect of adenosine is not reduced at high IDP concentrations. Pyrophosphate also inhibits the IDPase, but the effect is non-competitive and it is cumulative with that of ATP. We also observed that the latent activity of the IDPase (Triton-stimulated IDPase) is reduced by pre-treatment of the membranes with glutaraldehyde. The results indicate that Golgi IDPase is an allosteric enzyme which is positively modulated by IDP and negatively modulated by ATP and adenosine. Pyrophosphate inhibits the IDPase, but it seems to act at the catalytic site, whereas the other modulators appear to interact with a distinct regulatory site.