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. 1994 Feb 1;13(3):575–583. doi: 10.1002/j.1460-2075.1994.tb06295.x

Distinct structural elements of rab5 define its functional specificity.

H Stenmark 1, A Valencia 1, O Martinez 1, O Ullrich 1, B Goud 1, M Zerial 1
PMCID: PMC394846  PMID: 8313902

Abstract

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.

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Selected References

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

  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Antony C., Cibert C., Géraud G., Santa Maria A., Maro B., Mayau V., Goud B. The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach. J Cell Sci. 1992 Nov;103(Pt 3):785–796. doi: 10.1242/jcs.103.3.785. [DOI] [PubMed] [Google Scholar]
  3. Araki S., Kaibuchi K., Sasaki T., Hata Y., Takai Y. Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein. Mol Cell Biol. 1991 Mar;11(3):1438–1447. doi: 10.1128/mcb.11.3.1438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Araki S., Kikuchi A., Hata Y., Isomura M., Takai Y. Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J Biol Chem. 1990 Aug 5;265(22):13007–13015. [PubMed] [Google Scholar]
  5. Armstrong J., Craighead M. W., Watson R., Ponnambalam S., Bowden S. Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator. Mol Biol Cell. 1993 Jun;4(6):583–592. doi: 10.1091/mbc.4.6.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  8. Brennwald P., Novick P. Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4. Nature. 1993 Apr 8;362(6420):560–563. doi: 10.1038/362560a0. [DOI] [PubMed] [Google Scholar]
  9. Bucci C., Parton R. G., Mather I. H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. doi: 10.1016/0092-8674(92)90306-w. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Burton J., Roberts D., Montaldi M., Novick P., De Camilli P. A mammalian guanine-nucleotide-releasing protein enhances function of yeast secretory protein Sec4. Nature. 1993 Feb 4;361(6411):464–467. doi: 10.1038/361464a0. [DOI] [PubMed] [Google Scholar]
  12. Chavrier P., Gorvel J. P., Stelzer E., Simons K., Gruenberg J., Zerial M. Hypervariable C-terminal domain of rab proteins acts as a targeting signal. Nature. 1991 Oct 24;353(6346):769–772. doi: 10.1038/353769a0. [DOI] [PubMed] [Google Scholar]
  13. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  14. Conklin B. R., Bourne H. R. Structural elements of G alpha subunits that interact with G beta gamma, receptors, and effectors. Cell. 1993 May 21;73(4):631–641. doi: 10.1016/0092-8674(93)90245-l. [DOI] [PubMed] [Google Scholar]
  15. Dunn B., Stearns T., Botstein D. Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4. Nature. 1993 Apr 8;362(6420):563–565. doi: 10.1038/362563a0. [DOI] [PubMed] [Google Scholar]
  16. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gideon P., John J., Frech M., Lautwein A., Clark R., Scheffler J. E., Wittinghofer A. Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity. Mol Cell Biol. 1992 May;12(5):2050–2056. doi: 10.1128/mcb.12.5.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Goud B., Salminen A., Walworth N. C., Novick P. J. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  21. Goud B., Zahraoui A., Tavitian A., Saraste J. Small GTP-binding protein associated with Golgi cisternae. Nature. 1990 Jun 7;345(6275):553–556. doi: 10.1038/345553a0. [DOI] [PubMed] [Google Scholar]
  22. Hancock J. F., Cadwallader K., Marshall C. J. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. doi: 10.1002/j.1460-2075.1991.tb07992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haubruck H., Prange R., Vorgias C., Gallwitz D. The ras-related mouse ypt1 protein can functionally replace the YPT1 gene product in yeast. EMBO J. 1989 May;8(5):1427–1432. doi: 10.1002/j.1460-2075.1989.tb03524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  25. Khosravi-Far R., Lutz R. J., Cox A. D., Conroy L., Bourne J. R., Sinensky M., Balch W. E., Buss J. E., Der C. J. Isoprenoid modification of rab proteins terminating in CC or CXC motifs. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6264–6268. doi: 10.1073/pnas.88.14.6264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kinsella B. T., Erdman R. A., Maltese W. A. Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8934–8938. doi: 10.1073/pnas.88.20.8934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kinsella B. T., Maltese W. A. rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids. J Biol Chem. 1992 Feb 25;267(6):3940–3945. [PubMed] [Google Scholar]
  28. Lombardi D., Soldati T., Riederer M. A., Goda Y., Zerial M., Pfeffer S. R. Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J. 1993 Feb;12(2):677–682. doi: 10.1002/j.1460-2075.1993.tb05701.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Magee T., Newman C. The role of lipid anchors for small G proteins in membrane trafficking. Trends Cell Biol. 1992 Nov;2(11):318–323. doi: 10.1016/0962-8924(92)90172-j. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  32. Mistou M. Y., Jacquet E., Poullet P., Rensland H., Gideon P., Schlichting I., Wittinghofer A., Parmeggiani A. Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator. EMBO J. 1992 Jul;11(7):2391–2397. doi: 10.1002/j.1460-2075.1992.tb05303.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Oberhauser A. F., Monck J. R., Balch W. E., Fernandez J. M. Exocytotic fusion is activated by Rab3a peptides. Nature. 1992 Nov 19;360(6401):270–273. doi: 10.1038/360270a0. [DOI] [PubMed] [Google Scholar]
  35. Padfield P. J., Balch W. E., Jamieson J. D. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1656–1660. doi: 10.1073/pnas.89.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peter M., Chavrier P., Nigg E. A., Zerial M. Isoprenylation of rab proteins on structurally distinct cysteine motifs. J Cell Sci. 1992 Aug;102(Pt 4):857–865. doi: 10.1242/jcs.102.4.857. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Plutner H., Schwaninger R., Pind S., Balch W. E. Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway. EMBO J. 1990 Aug;9(8):2375–2383. doi: 10.1002/j.1460-2075.1990.tb07412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pryer N. K., Wuestehube L. J., Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. doi: 10.1146/annurev.bi.61.070192.002351. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  44. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Steele-Mortimer O., Clague M. J., Huber L. A., Chavrier P., Gruenberg J., Gorvel J. P. The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion. EMBO J. 1994 Jan 1;13(1):34–41. doi: 10.1002/j.1460-2075.1994.tb06232.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stouten P. F., Sander C., Wittinghofer A., Valencia A. How does the switch II region of G-domains work? FEBS Lett. 1993 Mar 29;320(1):1–6. doi: 10.1016/0014-5793(93)81644-f. [DOI] [PubMed] [Google Scholar]
  51. Strom M., Vollmer P., Tan T. J., Gallwitz D. A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family. Nature. 1993 Feb 25;361(6414):736–739. doi: 10.1038/361736a0. [DOI] [PubMed] [Google Scholar]
  52. Tisdale E. J., Bourne J. R., Khosravi-Far R., Der C. J., Balch W. E. GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992 Nov;119(4):749–761. doi: 10.1083/jcb.119.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Touchot N., Chardin P., Tavitian A. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8210–8214. doi: 10.1073/pnas.84.23.8210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Valencia A., Chardin P., Wittinghofer A., Sander C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 1991 May 14;30(19):4637–4648. doi: 10.1021/bi00233a001. [DOI] [PubMed] [Google Scholar]
  55. Verrotti A. C., Créchet J. B., Di Blasi F., Seidita G., Mirisola M. G., Kavounis C., Nastopoulos V., Burderi E., De Vendittis E., Parmeggiani A. RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP. EMBO J. 1992 Aug;11(8):2855–2862. doi: 10.1002/j.1460-2075.1992.tb05353.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Vriend G. WHAT IF: a molecular modeling and drug design program. J Mol Graph. 1990 Mar;8(1):52-6, 29. doi: 10.1016/0263-7855(90)80070-v. [DOI] [PubMed] [Google Scholar]
  57. Walworth N. C., Brennwald P., Kabcenell A. K., Garrett M., Novick P. Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2017–2028. doi: 10.1128/mcb.12.5.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wichmann H., Hengst L., Gallwitz D. Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p). Cell. 1992 Dec 24;71(7):1131–1142. doi: 10.1016/s0092-8674(05)80062-5. [DOI] [PubMed] [Google Scholar]
  59. Zerial M., Parton R., Chavrier P., Frank R. Localization of Rab family members in animal cells. Methods Enzymol. 1992;219:398–407. doi: 10.1016/0076-6879(92)19039-9. [DOI] [PubMed] [Google Scholar]
  60. Zerial M., Stenmark H. Rab GTPases in vesicular transport. Curr Opin Cell Biol. 1993 Aug;5(4):613–620. doi: 10.1016/0955-0674(93)90130-i. [DOI] [PubMed] [Google Scholar]
  61. van der Sluijs P., Hull M., Webster P., Mâle P., Goud B., Mellman I. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 1992 Sep 4;70(5):729–740. doi: 10.1016/0092-8674(92)90307-x. [DOI] [PubMed] [Google Scholar]

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