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. 1992 Nov 1;119(3):617–627. doi: 10.1083/jcb.119.3.617

Intracellular localization of the P21rho proteins

PMCID: PMC2289677  PMID: 1383236

Abstract

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha- ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

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Selected References

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  1. Abo A., Pick E., Hall A., Totty N., Teahan C. G., Segal A. W. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991 Oct 17;353(6345):668–670. doi: 10.1038/353668a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Braun U., Habermann B., Just I., Aktories K., Vandekerckhove J. Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product. FEBS Lett. 1989 Jan 16;243(1):70–76. doi: 10.1016/0014-5793(89)81220-7. [DOI] [PubMed] [Google Scholar]
  4. Béranger F., Goud B., Tavitian A., de Gunzburg J. Association of the Ras-antagonistic Rap1/Krev-1 proteins with the Golgi complex. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1606–1610. doi: 10.1073/pnas.88.5.1606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calés C., Hancock J. F., Marshall C. J., Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. doi: 10.1038/332548a0. [DOI] [PubMed] [Google Scholar]
  6. Chardin P., Boquet P., Madaule P., Popoff M. R., Rubin E. J., Gill D. M. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chardin P., Madaule P., Tavitian A. Coding sequence of human rho cDNAs clone 6 and clone 9. Nucleic Acids Res. 1988 Mar 25;16(6):2717–2717. doi: 10.1093/nar/16.6.2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Fukumoto Y., Kaibuchi K., Hori Y., Fujioka H., Araki S., Ueda T., Kikuchi A., Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. Oncogene. 1990 Sep;5(9):1321–1328. [PubMed] [Google Scholar]
  11. Garrett M. D., Self A. J., van Oers C., Hall A. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins. J Biol Chem. 1989 Jan 5;264(1):10–13. [PubMed] [Google Scholar]
  12. 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]
  13. Griffiths G., Back R., Marsh M. A quantitative analysis of the endocytic pathway in baby hamster kidney cells. J Cell Biol. 1989 Dec;109(6 Pt 1):2703–2720. doi: 10.1083/jcb.109.6.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hall A. Ras-related GTPases and the cytoskeleton. Mol Biol Cell. 1992 May;3(5):475–479. doi: 10.1091/mbc.3.5.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  16. Hancock J. F., Cadwallader K., Paterson H., Marshall C. J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 1991 Dec;10(13):4033–4039. doi: 10.1002/j.1460-2075.1991.tb04979.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  18. Hancock J. F., Marshall C. J., McKay I. A., Gardner S., Houslay M. D., Hall A., Wakelam M. J. Mutant but not normal p21 ras elevates inositol phospholipid breakdown in two different cell systems. Oncogene. 1988 Aug;3(2):187–193. [PubMed] [Google Scholar]
  19. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  20. Hopkins C. R. Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells. Cell. 1983 Nov;35(1):321–330. doi: 10.1016/0092-8674(83)90235-0. [DOI] [PubMed] [Google Scholar]
  21. Hori Y., Kikuchi A., Isomura M., Katayama M., Miura Y., Fujioka H., Kaibuchi K., Takai Y. Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins. Oncogene. 1991 Apr;6(4):515–522. [PubMed] [Google Scholar]
  22. Hoshijima M., Kondo J., Kikuchi A., Yamamoto K., Takai Y. Purification and characterization from bovine brain membranes of a GTP-binding protein with a Mr of 21,000, ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1--identification as the rhoA gene product. Brain Res Mol Brain Res. 1990 Jan;7(1):9–16. doi: 10.1016/0169-328x(90)90067-n. [DOI] [PubMed] [Google Scholar]
  23. Isomura M., Kaibuchi K., Yamamoto T., Kawamura S., Katayama M., Takai Y. Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol. Biochem Biophys Res Commun. 1990 Jun 15;169(2):652–659. doi: 10.1016/0006-291x(90)90380-6. [DOI] [PubMed] [Google Scholar]
  24. Isomura M., Kikuchi A., Ohga N., Takai Y. Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor. Oncogene. 1991 Jan;6(1):119–124. [PubMed] [Google Scholar]
  25. Katayama M., Kawata M., Yoshida Y., Horiuchi H., Yamamoto T., Matsuura Y., Takai Y. The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21. J Biol Chem. 1991 Jul 5;266(19):12639–12645. [PubMed] [Google Scholar]
  26. Kawahara Y., Kawata M., Sunako M., Araki S., Koide M., Tsuda T., Fukuzaki H., Takai Y. Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product. Biochem Biophys Res Commun. 1990 Jul 31;170(2):673–683. doi: 10.1016/0006-291x(90)92144-o. [DOI] [PubMed] [Google Scholar]
  27. Kim S., Kikuchi A., Mizoguchi A., Takai Y. Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain. Brain Res Mol Brain Res. 1989 Nov;6(2-3):167–176. doi: 10.1016/0169-328x(89)90051-x. [DOI] [PubMed] [Google Scholar]
  28. Knaus U. G., Heyworth P. G., Evans T., Curnutte J. T., Bokoch G. M. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991 Dec 6;254(5037):1512–1515. doi: 10.1126/science.1660188. [DOI] [PubMed] [Google Scholar]
  29. Lowe D. G., Goeddel D. V. Heterologous expression and characterization of the human R-ras gene product. Mol Cell Biol. 1987 Aug;7(8):2845–2856. doi: 10.1128/mcb.7.8.2845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Löwenadler B., Nilsson B., Abrahmsén L., Moks T., Ljungqvist L., Holmgren E., Paleus S., Josephson S., Philipson L., Uhlén M. Production of specific antibodies against protein A fusion proteins. EMBO J. 1986 Sep;5(9):2393–2398. doi: 10.1002/j.1460-2075.1986.tb04509.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Madaule P., Axel R. A novel ras-related gene family. Cell. 1985 May;41(1):31–40. doi: 10.1016/0092-8674(85)90058-3. [DOI] [PubMed] [Google Scholar]
  32. McCaffrey M., Johnson J. S., Goud B., Myers A. M., Rossier J., Popoff M. R., Madaule P., Boquet P. The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae. J Cell Biol. 1991 Oct;115(2):309–319. doi: 10.1083/jcb.115.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moores S. L., Schaber M. D., Mosser S. D., Rands E., O'Hara M. B., Garsky V. M., Marshall M. S., Pompliano D. L., Gibbs J. B. Sequence dependence of protein isoprenylation. J Biol Chem. 1991 Aug 5;266(22):14603–14610. [PubMed] [Google Scholar]
  34. Narumiya S., Sekine A., Fujiwara M. Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product. J Biol Chem. 1988 Nov 25;263(33):17255–17257. [PubMed] [Google Scholar]
  35. Olofsson B., Chardin P., Touchot N., Zahraoui A., Tavitian A. Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues. Oncogene. 1988 Aug;3(2):231–234. [PubMed] [Google Scholar]
  36. Paterson H. F., Self A. J., Garrett M. D., Just I., Aktories K., Hall A. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990 Sep;111(3):1001–1007. doi: 10.1083/jcb.111.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  38. Sahagian G. G., Neufeld E. F. Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. J Biol Chem. 1983 Jun 10;258(11):7121–7128. [PubMed] [Google Scholar]
  39. Schulz T. F., Vogetseder W., Mitterer M., Böck G., Johnson J. P., Dierich M. P. Individual epitopes of an 85,000 MW membrane adherence molecule are variably expressed on cells of different lineage. Immunology. 1988 Aug;64(4):581–586. [PMC free article] [PubMed] [Google Scholar]
  40. Stasia M. J., Jouan A., Bourmeyster N., Boquet P., Vignais P. V. ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility. Biochem Biophys Res Commun. 1991 Oct 31;180(2):615–622. doi: 10.1016/s0006-291x(05)81110-6. [DOI] [PubMed] [Google Scholar]
  41. Stoorvogel W., Geuze H. J., Griffith J. M., Schwartz A. L., Strous G. J. Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. J Cell Biol. 1989 Jun;108(6):2137–2148. doi: 10.1083/jcb.108.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Swanson J. A., Yirinec B. D., Silverstein S. C. Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J Cell Biol. 1985 Mar;100(3):851–859. doi: 10.1083/jcb.100.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Swanson J., Bushnell A., Silverstein S. C. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1921–1925. doi: 10.1073/pnas.84.7.1921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Toki C., Oda K., Ikehara Y. Demonstration of GTP-binding proteins and ADP-ribosylated proteins in rat liver Golgi fraction. Biochem Biophys Res Commun. 1989 Oct 16;164(1):333–338. doi: 10.1016/0006-291x(89)91722-1. [DOI] [PubMed] [Google Scholar]
  45. Ueda T., Kikuchi A., Ohga N., Yamamoto J., Takai Y. Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein. J Biol Chem. 1990 Jun 5;265(16):9373–9380. [PubMed] [Google Scholar]
  46. Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
  47. Yamamoto K., Kondo J., Hishida T., Teranishi Y., Takai Y. Purification and characterization of a GTP-binding protein with a molecular weight of 20,000 in bovine brain membranes. Identification as the rho gene product. J Biol Chem. 1988 Jul 15;263(20):9926–9932. [PubMed] [Google Scholar]

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