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Proc Natl Acad Sci U S A. 1993 Jul 15; 90(14): 6508–6512.

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.


Small GTP-binding proteins of the YPT/SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking. In mammals, Rab8 and Rab10 are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae. To study the localization of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus. The feasibility of this method was tested by using yeast SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-type SEC4, indicating that the modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection. In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the ruffling areas. In contrast, epitope-tagged Rab10 expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region. By light microscopy, the staining partially overlapped with that of a Golgi marker, beta-COP. Thus, despite the high degree homology of Rab8 and Rab10 (66% identity), the two proteins are localized to distinct cellular compartments. This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family.

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  • Novick P, Field C, Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. [PubMed]
  • Salminen A, Novick PJ. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. [PubMed]
  • 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. [PubMed]
  • Bourne HR. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. [PubMed]
  • Zahraoui A, Touchot N, Chardin P, Tavitian A. The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion. J Biol Chem. 1989 Jul 25;264(21):12394–12401. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Zahraoui A, Touchot N, Chardin P, Tavitian A. Complete coding sequences of the ras related rab 3 and 4 cDNAs. Nucleic Acids Res. 1988 Feb 11;16(3):1204–1204. [PMC free article] [PubMed]
  • Matsui Y, Kikuchi A, Kondo J, Hishida T, Teranishi Y, Takai Y. Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain. J Biol Chem. 1988 Aug 15;263(23):11071–11074. [PubMed]
  • Chavrier P, Simons K, Zerial M. The complexity of the Rab and Rho GTP-binding protein subfamilies revealed by a PCR cloning approach. Gene. 1992 Mar 15;112(2):261–264. [PubMed]
  • Chavrier P, Vingron M, Sander C, Simons K, Zerial M. Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol Cell Biol. 1990 Dec;10(12):6578–6585. [PMC free article] [PubMed]
  • Chavrier P, Parton RG, Hauri HP, 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. [PubMed]
  • Ngsee JK, Elferink LA, Scheller RH. A family of ras-like GTP-binding proteins expressed in electromotor neurons. J Biol Chem. 1991 Feb 5;266(4):2675–2680. [PubMed]
  • Elferink LA, Anzai K, Scheller RH. rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain. J Biol Chem. 1992 Mar 25;267(9):5768–5775. [PubMed]
  • Nagata K, Satoh T, Itoh H, Kozasa T, Okano Y, Doi T, Kaziro Y, Nozawa Y. The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library. FEBS Lett. 1990 Nov 26;275(1-2):29–32. [PubMed]
  • Goud B, Zahraoui A, Tavitian A, Saraste J. Small GTP-binding protein associated with Golgi cisternae. Nature. 1990 Jun 7;345(6275):553–556. [PubMed]
  • Baldini G, Hohl T, Lin HY, Lodish HF. Cloning of a Rab3 isotype predominantly expressed in adipocytes. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5049–5052. [PMC free article] [PubMed]
  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. [PubMed]
  • Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. [PubMed]
  • Rose MD, Broach JR. Cloning genes by complementation in yeast. Methods Enzymol. 1991;194:195–230. [PubMed]
  • Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature. 329(6142):840–842. [PubMed]
  • Nimmo ER, Sanders PG, Padua RA, Hughes D, Williamson R, Johnson KJ. The MEL gene: a new member of the RAB/YPT class of RAS-related genes. Oncogene. 1991 Aug;6(8):1347–1351. [PubMed]
  • Carlson M, Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. [PubMed]
  • Powell SK, Orci L, Craik CS, Moore HP. Efficient targeting to storage granules of human proinsulins with altered propeptide domain. J Cell Biol. 1988 Jun;106(6):1843–1851. [PMC free article] [PubMed]
  • Miller SG, Carnell L, Moore HH. Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling. J Cell Biol. 1992 Jul;118(2):267–283. [PMC free article] [PubMed]
  • Pagano RE, Sepanski MA, Martin OC. Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J Cell Biol. 1989 Nov;109(5):2067–2079. [PMC free article] [PubMed]
  • Molenaar CM, Prange R, Gallwitz D. A carboxyl-terminal cysteine residue is required for palmitic acid binding and biological activity of the ras-related yeast YPT1 protein. EMBO J. 1988 Apr;7(4):971–976. [PMC free article] [PubMed]
  • Walworth NC, Goud B, Kabcenell AK, Novick PJ. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. [PMC free article] [PubMed]
  • Duden R, Griffiths G, Frank R, Argos P, Kreis TE. Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell. 1991 Feb 8;64(3):649–665. [PubMed]
  • Donaldson JG, Lippincott-Schwartz J, Bloom GS, Kreis TE, Klausner RD. Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol. 1990 Dec;111(6 Pt 1):2295–2306. [PMC free article] [PubMed]
  • Lippincott-Schwartz J, Glickman J, Donaldson JG, Robbins J, Kreis TE, Seamon KB, Sheetz MP, Klausner RD. Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism. J Cell Biol. 1991 Feb;112(4):567–577. [PMC free article] [PubMed]
  • Reaves B, Banting G. Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. J Cell Biol. 1992 Jan;116(1):85–94. [PMC free article] [PubMed]
  • Vielh E, Touchot N, Zahraoui A, Tavitian A. Nucleotide sequence of a rat cDNA: rab1B, encoding a rab1-YPT related protein. Nucleic Acids Res. 1989 Feb 25;17(4):1770–1770. [PMC free article] [PubMed]
  • Sakurada K, Uchida K, Yamaguchi K, Aisaka K, Ito S, Ohmori T, Takeyama Y, Ueda T, Hori Y, Ohyanagi H, et al. Molecular cloning and characterization of a ras p21-like GTP-binding protein (24KG) from rat liver. Biochem Biophys Res Commun. 1991 Jun 28;177(3):1224–1232. [PubMed]
  • Fischer von Mollard G, Mignery GA, Baumert M, Perin MS, Hanson TJ, Burger PM, Jahn R, Südhof TC. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. [PMC free article] [PubMed]
  • Darchen F, Zahraoui A, Hammel F, Monteils MP, Tavitian A, Scherman D. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5692–5696. [PMC free article] [PubMed]
  • Matteoli M, Takei K, Cameron R, Hurlbut P, Johnston PA, Südhof TC, Jahn R, De Camilli P. Association of Rab3A with synaptic vesicles at late stages of the secretory pathway. J Cell Biol. 1991 Nov;115(3):625–633. [PMC free article] [PubMed]
  • Burgoyne RD, Morgan A. Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule. FEBS Lett. 1989 Mar 13;245(1-2):122–126. [PubMed]
  • Doucet JP, Fournier S, Parulekar M, Trifaró JM. Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells. FEBS Lett. 1989 Apr 10;247(1):127–131. [PubMed]
  • Philips MR, Abramson SB, Kolasinski SL, Haines KA, Weissmann G, Rosenfeld MG. Low molecular weight GTP-binding proteins in human neutrophil granule membranes. J Biol Chem. 1991 Jan 15;266(2):1289–1298. [PubMed]
  • van der Meulen J, Bhullar RP, Chancellor-Maddison KA. Association of a 24-kDa GTP-binding protein, Gn24, with human platelet alpha-granule membranes. FEBS Lett. 1991 Oct 7;291(1):122–126. [PubMed]
  • Rubins JB, Panchenko M, Shannon TM, Dickey BF. Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies. Am J Respir Cell Mol Biol. 1992 Mar;6(3):253–259. [PubMed]
  • Peterson JB. Small GTP-binding proteins associated with secretory vesicles of Paramecium. J Protozool. 1991 Sep-Oct;38(5):495–501. [PubMed]
  • Goud B, Salminen A, Walworth NC, Novick PJ. 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. [PubMed]
  • Haubruck H, Engelke U, Mertins P, Gallwitz D. Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue. EMBO J. 1990 Jun;9(6):1957–1962. [PMC free article] [PubMed]
  • Kinsella BT, Maltese WA. 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]
  • Chavrier P, Gorvel JP, 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. [PubMed]
  • Frech M, John J, Pizon V, Chardin P, Tavitian A, Clark R, McCormick F, Wittinghofer A. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science. 1990 Jul 13;249(4965):169–171. [PubMed]
  • MARCUS PI. Dynamics of surface modification in myxovirus-infected cells. Cold Spring Harb Symp Quant Biol. 1962;27:351–365. [PubMed]
  • Bergmann JE, Kupfer A, Singer SJ. Membrane insertion at the leading edge of motile fibroblasts. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1367–1371. [PMC free article] [PubMed]
  • Bretscher MS. Distribution of receptors for transferrin and low density lipoprotein on the surface of giant HeLa cells. Proc Natl Acad Sci U S A. 1983 Jan;80(2):454–458. [PMC free article] [PubMed]
  • Bretscher MS. Endocytosis and recycling of the fibronectin receptor in CHO cells. EMBO J. 1989 May;8(5):1341–1348. [PMC free article] [PubMed]
  • Myrdal SE, Auersperg N. p21ras. Heterogeneous localization in transformed cells. Exp Cell Res. 1985 Aug;159(2):441–450. [PubMed]
  • Bar-Sagi D, Suhan JP, McCormick F, Feramisco JR. Localization of phospholipase A2 in normal and ras-transformed cells. J Cell Biol. 1988 May;106(5):1649–1658. [PMC free article] [PubMed]

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