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We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, −2, and −3, while Munc-18c binds only to syntaxin-2 and −4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes. We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, −2, and −3, while Munc-18c binds only to syntaxin-2 and −4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes. INTRODUCTIONIn adipose tissue and muscle, the glucose transporter isoform 4, GLUT-4, is translocated from an intracellular vesicular pool to the cell surface in response to insulin (1Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 2Suzuki K. Kono T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2542-2545Crossref PubMed Scopus (768) Google Scholar), a process that plays a major role in whole body glucose homeostasis. To understand the molecular mechanisms governing this vesicular transport system, it will be necessary to identify and characterize the individual components of the trafficking machinery. The SNARE hypothesis (reviewed in Ref. 3Rothman J.E. Warren G. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google a for of vesicle and fusion in adipocytes. membrane protein membrane membrane attachment attachment protein of membrane the transport vesicle and syntaxin the membrane a which also the soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor This may the fusion of membrane the being by the of and the of the to in mammalian J.E. K. Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), three and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, E. T. PubMed Scopus Google Scholar, A. E. A. J. PubMed Scopus Google Scholar, A. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and three homologues E. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have a enriched in the membrane, binds with to which is in vesicles PubMed Scopus Google which to membrane between the and J.E. K. Full Text PDF PubMed Scopus Google Scholar), not with protein regulate the interaction between and of the the of by binding to the of in the fusion of transport vesicles with the plasma membrane S. 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L.J. A. Biol. PubMed Scopus Google these it was shown that syntaxin-4 is expressed in 3T3-L1 adipocytes, and that it is both in the plasma membrane and to a in intracellular a to that reported in the 3T3-L1 adipocytes, it was shown that a syntaxin-4 antibody a of glucose transport. the and that syntaxin-4 is involved in the of GLUT-4 trafficking in of insulin to an in cell surface of GLUT-4 is not that insulin of GLUT-4 from the intracellular with a in GLUT-4 J. S.W. J. Biol. Chem. Full Text PDF PubMed Google this an of insulin vesicle vesicle vesicle and fusion with the cell surface. it to be the of proteins in the study a for by This that a major of insulin is to the fusion of GLUT-4 vesicles with the cell surface. the both of these to be by a in potential the cell surface. this vesicles are observed the that fusion is in adipocytes and PubMed Scopus Google have to GLUT-4 vesicles that are the for of GLUT-4 the surface is vesicle that vesicle fusion is not E. S.W. J. Biol. Chem. Full Text PDF PubMed Google role by Munc-18c the interaction between syntaxin-4 and for insulin in Munc-18c may bind to syntaxin-4 and to bind to and so a vesicle may a by in syntaxin-4 thus the in of VAMP-2. This syntaxin-4 Munc-18c potential of insulin of a it shown that of Munc-18a by protein interaction with T. K. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google This a for it will be necessary to syntaxin-4 and Munc-18c in and an interaction is in the of The of Munc-18c and syntaxin-4 to the plasma membrane this of of Munc-18c syntaxin-4 was not specific to VAMP-2. also binds to that is expressed in adipocytes, and this interaction is also by Munc-18c the for these in adipocytes and cell is not from these it is thought that proteins are involved in the of a docking/fusion and of these the role for these We have shown that and are in intracellular vesicles in adipocytes S. J. J. Biol. PubMed Scopus Google Scholar), and so it is that the of these proteins is in their to regulate the of of This is because in the of insulin both and the cell surface of adipocytes, with the that both of these proteins and both of vesicles that are in A. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. J. Biol. PubMed Scopus Google this in of a in which insulin may modulate the of syntaxin-4 to bind of both and The role of Munc-18c the binding of syntaxin-4 to both and suggest a role a of this in this syntaxin-4 and Munc-18c may the and fusion of of and this is with their tissue distribution S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google INTRODUCTIONIn adipose tissue and muscle, the glucose transporter isoform 4, GLUT-4, is translocated from an intracellular vesicular pool to the cell surface in response to insulin (1Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 2Suzuki K. Kono T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2542-2545Crossref PubMed Scopus (768) Google Scholar), a process that plays a major role in whole body glucose homeostasis. To understand the molecular mechanisms governing this vesicular transport system, it will be necessary to identify and characterize the individual components of the trafficking machinery. The SNARE hypothesis (reviewed in Ref. 3Rothman J.E. Warren G. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google a for of vesicle and fusion in adipocytes. membrane protein membrane membrane attachment attachment protein of membrane the transport vesicle and syntaxin the membrane a which also the soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor This may the fusion of membrane the being by the of and the of the to in mammalian J.E. K. Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), three and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, E. T. PubMed Scopus Google Scholar, A. E. A. J. PubMed Scopus Google Scholar, A. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and three homologues E. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have a enriched in the membrane, binds with to which is in vesicles PubMed Scopus Google which to membrane between the and J.E. K. Full Text PDF PubMed Scopus Google Scholar), not with protein regulate the interaction between and of the the of by binding to the of in the fusion of transport vesicles with the plasma membrane S. Full Text PDF PubMed Scopus Google mammalian proteins, to have identified PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Munc-18a is expressed in the and with for binding to J. S. Full Text PDF PubMed Scopus Google of the of proteins in and their role in vesicle transport we out to identify proteins that may be involved in GLUT-4 trafficking in adipocytes. Munc-18 isoforms and identified S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), both of which the role of these isoforms in vesicle transport not and their syntaxin binding have not of in cell and their role in GLUT-4 only and A. E. A. J. PubMed Scopus Google E. S. T. J. Biol. Chem. Full Text PDF PubMed Google and A. E. A. J. PubMed Scopus Google Scholar, A. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of these isoforms with intracellular GLUT-4 GLUT-4, insulin-dependent movement to the cell surface A. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Using vesicles from adipocytes and an it shown that the of with of intracellular GLUT-4 in S. J. J. Biol. PubMed Scopus Google The GLUT-4, with the of to be to a the of which to be S. J. J. Biol. PubMed Scopus Google of these it is of interest to the for this may to insulin the of the study we that syntaxin-4 and Munc-18c may play a role in the trafficking of GLUT-4 in adipocytes. vitro show that Munc-18 isoforms syntaxin binding Munc-18c to of the previously identified in A. L.J. A. Biol. PubMed Scopus Google Scholar, J. A. A. J. PubMed Scopus Google and shown to bind to PubMed Scopus Google Using specific these proteins, we show that Munc-18c and syntaxin-4 have a similar subcellular distribution in 3T3-L1 adipocytes and both proteins insulin-dependent movement out of the intracellular membrane fraction that is enriched in GLUT-4. Microinjection of a antibody into 3T3-L1 adipocytes blocked movement of GLUT-4. These with the of Munc-18c to the interaction between syntaxin-4 and in these proteins in GLUT-4 trafficking in adipocytes.
The basic regularity of chromatin substructure that has been reported in rat liver chromatin (Hewish & Burgoyne, 1973b) was also detected in mouse chromatin. The regular series of DNA fragments produced by the action of Ca-Mg endonuclease on rat chromatin were studied further. The smallest single-stranded class has a molecular weight of approx. 45000-63000 and the smallest double-stranded class has a molecular weight of approx. 120000-150000. Studies of the substructure of the DNA fragments produced by the Ca-Mg endonuclease have shown that the regular series of double-stranded fragments have regular series of single-stranded fragments within them. It was concluded that the regular series of double-stranded fragments was probably a consequence of the regular series of single-stranded fragments. Digestion time-courses are presented for mouse and rat nuclear DNA.