Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 May;14(5):3459–3468. doi: 10.1128/mcb.14.5.3459

Cloning, characterization, and expression of a novel GDP dissociation inhibitor isoform from skeletal muscle.

A Shisheva 1, T C Südhof 1, M P Czech 1
PMCID: PMC358710  PMID: 7513052

Abstract

Cellular mechanisms for controlling membrane trafficking appear to involve small GTP-binding proteins such as the Rab proteins. Rab function is regulated by GDP dissociation inhibitor (GDI), which releases Rab proteins from membranes and inhibits GDP dissociation. Here we report the isolation of a full-length cDNA encoding a novel GDI isoform of 445 amino acids (GDI-2) with a deduced molecular weight of 50,649 from mouse skeletal muscle. Full-length and partial cDNA clones encoding a previously reported GDI protein (GDI-1) were also isolated from cDNA libraries prepared from rat brain and mouse skeletal muscle, respectively. The degree of deduced amino acid sequence identity between mouse GDI-2 and our mouse GDI-1 cDNA clone is 86%. Northern (RNA blot) analysis revealed that in human tissues, both GDI-1 and GDI-2 transcripts were abundant in brain, skeletal muscle, and pancreas but were weakly expressed in heart and liver. GDI-1 mRNA was expressed in kidney, whereas GDI-2 was almost absent, while in lung the relative amounts of these mRNA species were reversed. Specific antibodies against mouse GDI-1 and GDI-2 based on unique peptide sequences in the proteins were raised. Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes was accompanied by large increases in both mRNA and protein levels of GDI-1 and GDI-2. GDI-1 and GDI-2 expressed as glutathione S-transferase fusion proteins were both able to solubilize the membrane-bound forms of Rab4 and Rab5 in a GDP/GTP-dependent manner. Taken together, these data demonstrate that the protein products of at least two genes regulate the membrane dynamics of Rab proteins in mice.

Full text

PDF
3459

Images in this article

3465Image
on p.3465
3465Image
on p.3465
3465Image
on p.3465
3466Image
on p.3466
3466Image
on p.3466
3466Image
on p.3466
3467Image
on p.3467

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balch W. E. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. doi: 10.1016/0968-0004(90)90301-q. [DOI] [PubMed] [Google Scholar]
  2. Baldini G., Hohl T., Lin H. Y., Lodish H. F. Cloning of a Rab3 isotype predominantly expressed in adipocytes. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5049–5052. doi: 10.1073/pnas.89.11.5049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Becker J., Tan T. J., Trepte H. H., Gallwitz D. Mutational analysis of the putative effector domain of the GTP-binding Ypt1 protein in yeast suggests specific regulation by a novel GAP activity. EMBO J. 1991 Apr;10(4):785–792. doi: 10.1002/j.1460-2075.1991.tb08010.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burstein E. S., Linko-Stentz K., Lu Z. J., Macara I. G. Regulation of the GTPase activity of the ras-like protein p25rab3A. Evidence for a rab3A-specific GAP. J Biol Chem. 1991 Feb 15;266(5):2689–2692. [PubMed] [Google Scholar]
  5. Burstein E. S., Macara I. G. Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1154–1158. doi: 10.1073/pnas.89.4.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cormont M., Tanti J. F., Zahraoui A., Van Obberghen E., Tavitian A., Le Marchand-Brustel Y. Insulin and okadaic acid induce Rab4 redistribution in adipocytes. J Biol Chem. 1993 Sep 15;268(26):19491–19497. [PubMed] [Google Scholar]
  8. Czech M. P., Buxton J. M. Insulin action on the internalization of the GLUT4 glucose transporter in isolated rat adipocytes. J Biol Chem. 1993 May 5;268(13):9187–9190. [PubMed] [Google Scholar]
  9. Czech M. P., Clancy B. M., Pessino A., Woon C. W., Harrison S. A. Complex regulation of simple sugar transport in insulin-responsive cells. Trends Biochem Sci. 1992 May;17(5):197–201. doi: 10.1016/0968-0004(92)90266-c. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorvel J. P., Chavrier P., Zerial M., Gruenberg J. rab5 controls early endosome fusion in vitro. Cell. 1991 Mar 8;64(5):915–925. doi: 10.1016/0092-8674(91)90316-q. [DOI] [PubMed] [Google Scholar]
  13. Harrison S. A., Buxton J. M., Clancy B. M., Czech M. P. Insulin regulation of hexose transport in mouse 3T3-L1 cells expressing the human HepG2 glucose transporter. J Biol Chem. 1990 Nov 25;265(33):20106–20116. [PubMed] [Google Scholar]
  14. Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
  15. Jhun B. H., Rampal A. L., Liu H., Lachaal M., Jung C. Y. Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling. J Biol Chem. 1992 Sep 5;267(25):17710–17715. [PubMed] [Google Scholar]
  16. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lledo P. M., Vernier P., Vincent J. D., Mason W. T., Zorec R. Inhibition of Rab3B expression attenuates Ca(2+)-dependent exocytosis in rat anterior pituitary cells. Nature. 1993 Aug 5;364(6437):540–544. doi: 10.1038/364540a0. [DOI] [PubMed] [Google Scholar]
  18. Matsui Y., Kikuchi A., Araki S., Hata Y., Kondo J., Teranishi Y., Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. Mol Cell Biol. 1990 Aug;10(8):4116–4122. doi: 10.1128/mcb.10.8.4116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moya M., Roberts D., Novick P. DSS4-1 is a dominant suppressor of sec4-8 that encodes a nucleotide exchange protein that aids Sec4p function. Nature. 1993 Feb 4;361(6411):460–463. doi: 10.1038/361460a0. [DOI] [PubMed] [Google Scholar]
  20. Nonaka H., Kaibuchi K., Shimizu K., Yamamoto J., Takai Y. Tissue and subcellular distributions of an inhibitory GDP/GTP exchange protein (GDI) for smg p25A by use of its antibody. Biochem Biophys Res Commun. 1991 Jan 31;174(2):556–563. doi: 10.1016/0006-291x(91)91453-j. [DOI] [PubMed] [Google Scholar]
  21. Pfeffer S. R. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. doi: 10.1016/0962-8924(92)90161-f. [DOI] [PubMed] [Google Scholar]
  22. Plutner H., Cox A. D., Pind S., Khosravi-Far R., Bourne J. R., Schwaninger R., Der C. J., Balch W. E. Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J Cell Biol. 1991 Oct;115(1):31–43. doi: 10.1083/jcb.115.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Regazzi R., Kikuchi A., Takai Y., Wollheim C. B. The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J Biol Chem. 1992 Sep 5;267(25):17512–17519. [PubMed] [Google Scholar]
  24. Rexach M. F., Schekman R. W. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219–229. doi: 10.1083/jcb.114.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sasaki T., Kaibuchi K., Kabcenell A. K., Novick P. J., Takai Y. A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein. Mol Cell Biol. 1991 May;11(5):2909–2912. doi: 10.1128/mcb.11.5.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sasaki T., Kikuchi A., Araki S., Hata Y., Isomura M., Kuroda S., Takai Y. Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem. 1990 Feb 5;265(4):2333–2337. [PubMed] [Google Scholar]
  29. Segev N. Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein. Science. 1991 Jun 14;252(5012):1553–1556. doi: 10.1126/science.1904626. [DOI] [PubMed] [Google Scholar]
  30. Shirataki H., Kaibuchi K., Sakoda T., Kishida S., Yamaguchi T., Wada K., Miyazaki M., Takai Y. Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol Cell Biol. 1993 Apr;13(4):2061–2068. doi: 10.1128/mcb.13.4.2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shisheva A., Shechter Y. Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect. Endocrinology. 1993 Oct;133(4):1562–1568. doi: 10.1210/endo.133.4.8404595. [DOI] [PubMed] [Google Scholar]
  32. Soldati T., Riederer M. A., Pfeffer S. R. Rab GDI: a solubilizing and recycling factor for rab9 protein. Mol Biol Cell. 1993 Apr;4(4):425–434. doi: 10.1091/mbc.4.4.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Takai Y., Kaibuchi K., Kikuchi A., Kawata M. Small GTP-binding proteins. Int Rev Cytol. 1992;133:187–230. doi: 10.1016/s0074-7696(08)61861-6. [DOI] [PubMed] [Google Scholar]
  34. Ueda T., Takeyama Y., Ohmori T., Ohyanagi H., Saitoh Y., Takai Y. Purification and characterization from rat liver cytosol of a GDP dissociation inhibitor (GDI) for liver 24K G, a ras p21-like GTP-binding protein, with properties similar to those of smg p25A GDI. Biochemistry. 1991 Jan 29;30(4):909–917. doi: 10.1021/bi00218a005. [DOI] [PubMed] [Google Scholar]
  35. Ullrich O., Stenmark H., Alexandrov K., Huber L. A., Kaibuchi K., Sasaki T., Takai Y., Zerial M. Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins. J Biol Chem. 1993 Aug 25;268(24):18143–18150. [PubMed] [Google Scholar]
  36. Ushkaryov Y. A., Südhof T. C. Neurexin III alpha: extensive alternative splicing generates membrane-bound and soluble forms. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6410–6414. doi: 10.1073/pnas.90.14.6410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yang J., Holman G. D. Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem. 1993 Mar 5;268(7):4600–4603. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES