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. 1998 Aug 1;333(Pt 3):497–504. doi: 10.1042/bj3330497

Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases.

A L Wilson 1, R A Erdman 1, F Castellano 1, W A Maltese 1
PMCID: PMC1219609  PMID: 9677305

Abstract

Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 prenylation in vivo, the incorporation of [3H]mevalonate into Rab8 was measured in human embryonal kidney 293 cells under conditions where the activity of GGTaseI, but not GGTaseII, was blocked by the peptidomimetic inhibitor GGTI-298. The GGTaseI inhibitor did not prevent prenylation of either overexpressed Myc-tagged Rab8 or endogenous Rab8, whereas prenylation of a known GGTaseI substrate with the same carboxyl-terminal motif, Cdc42Hs, was completely blocked. To rule out the possibility that the apparent prenylation of Rab8 by GGTaseII occurs only when GGTaseI activity is eliminated, metabolic labelling studies were carried out in the absence of the GGTaseI inhibitor, using a REP-binding-deficient Rab8 construct (Y78D) that cannot serve as a substrate for GGTaseII, but is indistinguishable from wild-type Rab8 as a substrate for GGTaseI. Prenylation of the Y78D mutant was reduced by 60-70% in intact cells, consistent with the conclusion that the majority of Rab8 is prenylated by the REP/GGTaseII system in vivo.

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Selected References

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  1. Alberts A. W., Chen J., Kuron G., Hunt V., Huff J., Hoffman C., Rothrock J., Lopez M., Joshua H., Harris E. Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3957–3961. doi: 10.1073/pnas.77.7.3957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexandrov K., Horiuchi H., Steele-Mortimer O., Seabra M. C., Zerial M. Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes. EMBO J. 1994 Nov 15;13(22):5262–5273. doi: 10.1002/j.1460-2075.1994.tb06860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andersson S., Davis D. L., Dahlbäck H., Jörnvall H., Russell D. W. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed] [Google Scholar]
  4. Andres D. A., Seabra M. C., Brown M. S., Armstrong S. A., Smeland T. E., Cremers F. P., Goldstein J. L. cDNA cloning of component A of Rab geranylgeranyl transferase and demonstration of its role as a Rab escort protein. Cell. 1993 Jun 18;73(6):1091–1099. doi: 10.1016/0092-8674(93)90639-8. [DOI] [PubMed] [Google Scholar]
  5. Armstrong S. A., Seabra M. C., Südhof T. C., Goldstein J. L., Brown M. S. cDNA cloning and expression of the alpha and beta subunits of rat Rab geranylgeranyl transferase. J Biol Chem. 1993 Jun 5;268(16):12221–12229. [PubMed] [Google Scholar]
  6. Beranger F., Cadwallader K., Porfiri E., Powers S., Evans T., de Gunzburg J., Hancock J. F. Determination of structural requirements for the interaction of Rab6 with RabGDI and Rab geranylgeranyltransferase. J Biol Chem. 1994 May 6;269(18):13637–13643. [PubMed] [Google Scholar]
  7. Casey P. J., Seabra M. C. Protein prenyltransferases. J Biol Chem. 1996 Mar 8;271(10):5289–5292. doi: 10.1074/jbc.271.10.5289. [DOI] [PubMed] [Google Scholar]
  8. Casey P. J., Thissen J. A., Moomaw J. F. Enzymatic modification of proteins with a geranylgeranyl isoprenoid. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8631–8635. doi: 10.1073/pnas.88.19.8631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Farnsworth C. C., Seabra M. C., Ericsson L. H., Gelb M. H., Glomset J. A. Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11963–11967. doi: 10.1073/pnas.91.25.11963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Joberty G., Tavitian A., Zahraoui A. Isoprenylation of Rab proteins possessing a C-terminal CaaX motif. FEBS Lett. 1993 Sep 20;330(3):323–328. doi: 10.1016/0014-5793(93)80897-4. [DOI] [PubMed] [Google Scholar]
  12. Khosravi-Far R., Clark G. J., Abe K., Cox A. D., McLain T., Lutz R. J., Sinensky M., Der C. J. Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct. J Biol Chem. 1992 Dec 5;267(34):24363–24368. [PubMed] [Google Scholar]
  13. 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]
  14. Kinsella B. T., Maltese W. A. rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif. J Biol Chem. 1991 May 5;266(13):8540–8544. [PubMed] [Google Scholar]
  15. 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]
  16. Krupinski J., Lehman T. C., Frankenfield C. D., Zwaagstra J. C., Watson P. A. Molecular diversity in the adenylylcyclase family. Evidence for eight forms of the enzyme and cloning of type VI. J Biol Chem. 1992 Dec 5;267(34):24858–24862. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Madaule P., Axel R., Myers A. M. Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Feb;84(3):779–783. doi: 10.1073/pnas.84.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Musha T., Kawata M., Takai Y. The geranylgeranyl moiety but not the methyl moiety of the smg-25A/rab3A protein is essential for the interactions with membrane and its inhibitory GDP/GTP exchange protein. J Biol Chem. 1992 May 15;267(14):9821–9825. [PubMed] [Google Scholar]
  21. Novick P., Brennwald P. Friends and family: the role of the Rab GTPases in vesicular traffic. Cell. 1993 Nov 19;75(4):597–601. doi: 10.1016/0092-8674(93)90478-9. [DOI] [PubMed] [Google Scholar]
  22. Nuoffer C., Balch W. E. GTPases: multifunctional molecular switches regulating vesicular traffic. Annu Rev Biochem. 1994;63:949–990. doi: 10.1146/annurev.bi.63.070194.004505. [DOI] [PubMed] [Google Scholar]
  23. Overmeyer J. H., Wilson A. L., Erdman R. A., Maltese W. A. The putative "switch 2" domain of the Ras-related GTPase, Rab1B, plays an essential role in the interaction with Rab escort protein. Mol Biol Cell. 1998 Jan;9(1):223–235. doi: 10.1091/mbc.9.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Pfeffer S. R., Dirac-Svejstrup A. B., Soldati T. Rab GDP dissociation inhibitor: putting rab GTPases in the right place. J Biol Chem. 1995 Jul 21;270(29):17057–17059. doi: 10.1074/jbc.270.29.17057. [DOI] [PubMed] [Google Scholar]
  26. Pfeffer S. R. Rab GTPases: master regulators of membrane trafficking. Curr Opin Cell Biol. 1994 Aug;6(4):522–526. doi: 10.1016/0955-0674(94)90071-x. [DOI] [PubMed] [Google Scholar]
  27. Reiss Y., Goldstein J. L., Seabra M. C., Casey P. J., Brown M. S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell. 1990 Jul 13;62(1):81–88. doi: 10.1016/0092-8674(90)90242-7. [DOI] [PubMed] [Google Scholar]
  28. Reiss Y., Stradley S. J., Gierasch L. M., Brown M. S., Goldstein J. L. Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):732–736. doi: 10.1073/pnas.88.3.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Repko E. M., Maltese W. A. Post-translational isoprenylation of cellular proteins is altered in response to mevalonate availability. J Biol Chem. 1989 Jun 15;264(17):9945–9952. [PubMed] [Google Scholar]
  30. Rowell C. A., Kowalczyk J. J., Lewis M. D., Garcia A. M. Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo. J Biol Chem. 1997 May 30;272(22):14093–14097. doi: 10.1074/jbc.272.22.14093. [DOI] [PubMed] [Google Scholar]
  31. Sanford J. C., Pan Y., Wessling-Resnick M. Prenylation of Rab5 is dependent on guanine nucleotide binding. J Biol Chem. 1993 Nov 15;268(32):23773–23776. [PubMed] [Google Scholar]
  32. Sanford J. C., Pan Y., Wessling-Resnick M. Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines. Mol Biol Cell. 1995 Jan;6(1):71–85. doi: 10.1091/mbc.6.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schalk I., Zeng K., Wu S. K., Stura E. A., Matteson J., Huang M., Tandon A., Wilson I. A., Balch W. E. Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature. 1996 May 2;381(6577):42–48. doi: 10.1038/381042a0. [DOI] [PubMed] [Google Scholar]
  34. Schiedel A. C., Barnekow A., Mayer T. Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells. FEBS Lett. 1995 Nov 27;376(1-2):113–119. doi: 10.1016/0014-5793(95)01258-0. [DOI] [PubMed] [Google Scholar]
  35. Seabra M. C., Brown M. S., Slaughter C. A., Südhof T. C., Goldstein J. L. Purification of component A of Rab geranylgeranyl transferase: possible identity with the choroideremia gene product. Cell. 1992 Sep 18;70(6):1049–1057. doi: 10.1016/0092-8674(92)90253-9. [DOI] [PubMed] [Google Scholar]
  36. Seabra M. C., Goldstein J. L., Südhof T. C., Brown M. S. Rab geranylgeranyl transferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys. J Biol Chem. 1992 Jul 15;267(20):14497–14503. [PubMed] [Google Scholar]
  37. Seabra M. C. Nucleotide dependence of Rab geranylgeranylation. Rab escort protein interacts preferentially with GDP-bound Rab. J Biol Chem. 1996 Jun 14;271(24):14398–14404. doi: 10.1074/jbc.271.24.14398. [DOI] [PubMed] [Google Scholar]
  38. Seabra M. C., Reiss Y., Casey P. J., Brown M. S., Goldstein J. L. Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell. 1991 May 3;65(3):429–434. doi: 10.1016/0092-8674(91)90460-g. [DOI] [PubMed] [Google Scholar]
  39. Shen F., Seabra M. C. Mechanism of digeranylgeranylation of Rab proteins. Formation of a complex between monogeranylgeranyl-Rab and Rab escort protein. J Biol Chem. 1996 Feb 16;271(7):3692–3698. doi: 10.1074/jbc.271.7.3692. [DOI] [PubMed] [Google Scholar]
  40. Shinjo K., Koland J. G., Hart M. J., Narasimhan V., Johnson D. I., Evans T., Cerione R. A. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9853–9857. doi: 10.1073/pnas.87.24.9853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Stenmark H., Valencia A., Martinez O., Ullrich O., Goud B., Zerial M. Distinct structural elements of rab5 define its functional specificity. EMBO J. 1994 Feb 1;13(3):575–583. doi: 10.1002/j.1460-2075.1994.tb06295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Vogt A., Qian Y., McGuire T. F., Hamilton A. D., Sebti S. M. Protein geranylgeranylation, not farnesylation, is required for the G1 to S phase transition in mouse fibroblasts. Oncogene. 1996 Nov 7;13(9):1991–1999. [PubMed] [Google Scholar]
  45. Whyte D. B., Kirschmeier P., Hockenberry T. N., Nunez-Oliva I., James L., Catino J. J., Bishop W. R., Pai J. K. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem. 1997 May 30;272(22):14459–14464. doi: 10.1074/jbc.272.22.14459. [DOI] [PubMed] [Google Scholar]
  46. Wilson A. L., Erdman R. A., Maltese W. A. Association of Rab1B with GDP-dissociation inhibitor (GDI) is required for recycling but not initial membrane targeting of the Rab protein. J Biol Chem. 1996 May 3;271(18):10932–10940. doi: 10.1074/jbc.271.18.10932. [DOI] [PubMed] [Google Scholar]
  47. Wilson A. L., Maltese W. A. Isoprenylation of Rab1B is impaired by mutations in its effector domain. J Biol Chem. 1993 Jul 15;268(20):14561–14564. [PubMed] [Google Scholar]
  48. Wilson A. L., Sheridan K. M., Erdman R. A., Maltese W. A. Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells. Biochem J. 1996 Sep 15;318(Pt 3):1007–1014. doi: 10.1042/bj3181007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yokoyama K., Goodwin G. W., Ghomashchi F., Glomset J. A., Gelb M. H. A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5302–5306. doi: 10.1073/pnas.88.12.5302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Zhang F. L., Kirschmeier P., Carr D., James L., Bond R. W., Wang L., Patton R., Windsor W. T., Syto R., Zhang R. Characterization of Ha-ras, N-ras, Ki-Ras4A, and Ki-Ras4B as in vitro substrates for farnesyl protein transferase and geranylgeranyl protein transferase type I. J Biol Chem. 1997 Apr 11;272(15):10232–10239. doi: 10.1074/jbc.272.15.10232. [DOI] [PubMed] [Google Scholar]
  52. Zhang F. L., Moomaw J. F., Casey P. J. Properties and kinetic mechanism of recombinant mammalian protein geranylgeranyltransferase type I. J Biol Chem. 1994 Sep 23;269(38):23465–23470. [PubMed] [Google Scholar]

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