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J Cell Biol. 1994 Jul 1; 126(1): 87–97.
PMCID: PMC2120106

G-protein ligands inhibit in vitro reactions of vacuole inheritance


During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469- 1479). We now report a simple and reliable assay to quantify vacuole-to- vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2- 1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP- hydrolyzing G proteins may play a key role in the in vitro fusion events.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Achstetter T, Emter O, Ehmann C, Wolf DH. Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast. J Biol Chem. 1984 Nov 10;259(21):13334–13343. [PubMed]
  • Ammerer G, Hunter CP, Rothman JH, Saari GC, Valls LA, Stevens TH. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol. 1986 Jul;6(7):2490–2499. [PMC free article] [PubMed]
  • Anraku Y, Umemoto N, Hirata R, Wada Y. Structure and function of the yeast vacuolar membrane proton ATPase. J Bioenerg Biomembr. 1989 Oct;21(5):589–603. [PubMed]
  • Baker D, Hicke L, Rexach M, Schleyer M, Schekman R. Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell. 1988 Jul 29;54(3):335–344. [PubMed]
  • Balch WE, Dunphy WG, Braell WA, Rothman JE. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. [PubMed]
  • Barlowe C, d'Enfert C, Schekman R. Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum. J Biol Chem. 1993 Jan 15;268(2):873–879. [PubMed]
  • Beckers CJ, Balch WE. Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol. 1989 Apr;108(4):1245–1256. [PMC free article] [PubMed]
  • Bigay J, Deterre P, Pfister C, Chabre M. Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP. EMBO J. 1987 Oct;6(10):2907–2913. [PMC free article] [PubMed]
  • Birky CW., Jr The partitioning of cytoplasmic organelles at cell division. Int Rev Cytol Suppl. 1983;15:49–89. [PubMed]
  • Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. [PubMed]
  • Boman AL, Delannoy MR, Wilson KL. GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. J Cell Biol. 1992 Jan;116(2):281–294. [PMC free article] [PubMed]
  • Bomsel M, Mostov K. Role of heterotrimeric G proteins in membrane traffic. Mol Biol Cell. 1992 Dec;3(12):1317–1328. [PMC free article] [PubMed]
  • Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. [PubMed]
  • Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7972–7976. [PMC free article] [PubMed]
  • Burgoyne RD. Phosphoinositides in vesicular traffic. Trends Biochem Sci. 1994 Feb;19(2):55–57. [PubMed]
  • Carney DH, Scott DL, Gordon EA, LaBelle EF. Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation. Cell. 1985 Sep;42(2):479–488. [PubMed]
  • Carter LL, Redelmeier TE, Woollenweber LA, Schmid SL. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J Cell Biol. 1993 Jan;120(1):37–45. [PMC free article] [PubMed]
  • Clague MJ, Urbé S, Aniento F, Gruenberg J. Vacuolar ATPase activity is required for endosomal carrier vesicle formation. J Biol Chem. 1994 Jan 7;269(1):21–24. [PubMed]
  • Colombo MI, Mayorga LS, Casey PJ, Stahl PD. Evidence of a role for heterotrimeric GTP-binding proteins in endosome fusion. Science. 1992 Mar 27;255(5052):1695–1697. [PubMed]
  • Conradt B, Shaw J, Vida T, Emr S, Wickner W. In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol. 1992 Dec;119(6):1469–1479. [PMC free article] [PubMed]
  • Conradt B, Haas A, Wickner W. Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro. J Cell Biol. 1994 Jul;126(1):99–110. [PMC free article] [PubMed]
  • Dietzel C, Kurjan J. The yeast SCG1 gene: a G alpha-like protein implicated in the a- and alpha-factor response pathway. Cell. 1987 Sep 25;50(7):1001–1010. [PubMed]
  • Fischer T, Bronner C, Landry Y, Mousli M. The mechanism of inhibition of alkylamines on the mast-cell peptidergic pathway. Biochim Biophys Acta. 1993 Apr 16;1176(3):305–312. [PubMed]
  • Gabev E, Kasianowicz J, Abbott T, McLaughlin S. Binding of neomycin to phosphatidylinositol 4,5-bisphosphate (PIP2). Biochim Biophys Acta. 1989 Feb 13;979(1):105–112. [PubMed]
  • Gomes de Mesquita DS, ten Hoopen R, Woldringh CL. Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle. J Gen Microbiol. 1991 Oct;137(10):2447–2454. [PubMed]
  • Gruenberg J, Clague MJ. Regulation of intracellular membrane transport. Curr Opin Cell Biol. 1992 Aug;4(4):593–599. [PubMed]
  • Higashijima T, Burnier J, Ross EM. Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. J Biol Chem. 1990 Aug 25;265(24):14176–14186. [PubMed]
  • Hirai Y, Yasuhara T, Yoshida H, Nakajima T, Fujino M, Kitada C. A new mast cell degranulating peptide "mastoparan" in the venom of Vespula lewisii. Chem Pharm Bull (Tokyo) 1979 Aug;27(8):1942–1944. [PubMed]
  • Ho WC, Allan VJ, van Meer G, Berger EG, Kreis TE. Reclustering of scattered Golgi elements occurs along microtubules. Eur J Cell Biol. 1989 Apr;48(2):250–263. [PubMed]
  • Jones EW, Zubenko GS, Parker RR. PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae. Genetics. 1982 Dec;102(4):665–677. [PMC free article] [PubMed]
  • Jones DT, Masters SB, Bourne HR, Reed RR. Biochemical characterization of three stimulatory GTP-binding proteins. The large and small forms of Gs and the olfactory-specific G-protein, Golf. J Biol Chem. 1990 Feb 15;265(5):2671–2676. [PubMed]
  • Kaneko Y, Hayashi N, Toh-e A, Banno I, Oshima Y. Structural characteristics of the PHO8 gene encoding repressible alkaline phosphatase in Saccharomyces cerevisiae. Gene. 1987;58(1):137–148. [PubMed]
  • Kaneko Y, Toh-e A, Banno I, Oshima Y. Molecular characterization of a specific p-nitrophenylphosphatase gene, PHO13, and its mapping by chromosome fragmentation in Saccharomyces cerevisiae. Mol Gen Genet. 1989 Dec;220(1):133–139. [PubMed]
  • Katsu T, Kuroko M, Morikawa T, Sanchika K, Yamanaka H, Shinoda S, Fujita Y. Interaction of wasp venom mastoparan with biomembranes. Biochim Biophys Acta. 1990 Aug 24;1027(2):185–190. [PubMed]
  • Klionsky DJ, Emr SD. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 1989 Aug;8(8):2241–2250. [PMC free article] [PubMed]
  • Klionsky DJ, Herman PK, Emr SD. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. [PMC free article] [PubMed]
  • Koch G, Haberman B, Mohr C, Just I, Aktories K. Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac. FEBS Lett. 1991 Oct 21;291(2):336–340. [PubMed]
  • Kopf GS, Woolkalis MJ. ADP-ribosylation of G proteins with pertussis toxin. Methods Enzymol. 1991;195:257–266. [PubMed]
  • Leyte A, Barr FA, Kehlenbach RH, Huttner WB. Multiple trimeric G-proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation. EMBO J. 1992 Dec;11(13):4795–4804. [PMC free article] [PubMed]
  • Lodhi S, Weiner ND, Schacht J. Interactions of neomycin with monomolecular films of polyphosphoinositides and other lipids. Biochim Biophys Acta. 1979 Oct 19;557(1):1–8. [PubMed]
  • Lucocq JM, Warren G. Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells. EMBO J. 1987 Nov;6(11):3239–3246. [PMC free article] [PubMed]
  • Malencik DA, Anderson SR. High affinity binding of the mastoparans by calmodulin. Biochem Biophys Res Commun. 1983 Jul 18;114(1):50–56. [PubMed]
  • Marche P, Koutouzov S, Girard A. Impairment of membrane phosphoinositide metabolism by aminoglycoside antibiotics: streptomycin, amikacin, kanamycin, dibekacin, gentamicin and neomycin. J Pharmacol Exp Ther. 1983 Nov;227(2):415–420. [PubMed]
  • Matteoni R, Kreis TE. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. [PMC free article] [PubMed]
  • Mayorga LS, Diaz R, Stahl PD. Regulatory role for GTP-binding proteins in endocytosis. Science. 1989 Jun 23;244(4911):1475–1477. [PubMed]
  • Mayorga LS, Diaz R, Colombo MI, Stahl PD. GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion. Cell Regul. 1989 Nov;1(1):113–124. [PMC free article] [PubMed]
  • McDonald LJ, Wainschel LA, Oppenheimer NJ, Moss J. Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction. Biochemistry. 1992 Dec 1;31(47):11881–11887. [PubMed]
  • Melançon P, Glick BS, Malhotra V, Weidman PJ, Serafini T, Gleason ML, Orci L, Rothman JE. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. [PubMed]
  • Mitchell JK, Fonzi WA, Wilkerson J, Opheim DJ. A particulate form of alkaline phosphatase in the yeast, Saccharomyces cerevisiae. Biochim Biophys Acta. 1981 Feb 13;657(2):482–494. [PubMed]
  • Mousli M, Bueb JL, Bronner C, Rouot B, Landry Y. G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides. Trends Pharmacol Sci. 1990 Sep;11(9):358–362. [PubMed]
  • Nakafuku M, Itoh H, Nakamura S, Kaziro Y. Occurrence in Saccharomyces cerevisiae of a gene homologous to the cDNA coding for the alpha subunit of mammalian G proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2140–2144. [PMC free article] [PubMed]
  • Nakafuku M, Obara T, Kaibuchi K, Miyajima I, Miyajima A, Itoh H, Nakamura S, Arai K, Matsumoto K, Kaziro Y. Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1374–1378. [PMC free article] [PubMed]
  • Obara T, Nakafuku M, Yamamoto M, Kaziro Y. Isolation and characterization of a gene encoding a G-protein alpha subunit from Schizosaccharomyces pombe: involvement in mating and sporulation pathways. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5877–5881. [PMC free article] [PubMed]
  • Oppi C, Wagner T, Crisari A, Camerini B, Tocchini Valentini GP. Attenuation of GTPase activity of recombinant G(o) alpha by peptides representing sequence permutations of mastoparan. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8268–8272. [PMC free article] [PubMed]
  • Paris S, Eckstein F. Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5'-[beta-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp. Biochem J. 1992 Jun 1;284(Pt 2):327–332. [PMC free article] [PubMed]
  • Pfeffer SR. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. [PubMed]
  • Pimplikar SW, Simons K. Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature. 1993 Apr 1;362(6419):456–458. [PubMed]
  • Prentki M, Deeney JT, Matschinsky FM, Joseph SK. Neomycin: a specific drug to study the inositol-phospholipid signalling system? FEBS Lett. 1986 Mar 3;197(1-2):285–288. [PubMed]
  • Pryer NK, Wuestehube LJ, Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. [PubMed]
  • Raymond CK, Roberts CJ, Moore KE, Howald I, Stevens TH. Biogenesis of the vacuole in Saccharomyces cerevisiae. Int Rev Cytol. 1992;139:59–120. [PubMed]
  • Read GW, Kiefer EF. Benzalkonium chloride: selective inhibitor of histamine release induced by compound 48/80 and other polyamines. J Pharmacol Exp Ther. 1979 Dec;211(3):711–715. [PubMed]
  • Rexach MF, Schekman RW. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219–229. [PMC free article] [PubMed]
  • Rothman JE, Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. [PubMed]
  • Ruohola H, Kabcenell AK, Ferro-Novick S. Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant. J Cell Biol. 1988 Oct;107(4):1465–1476. [PMC free article] [PubMed]
  • Schwaninger R, Plutner H, Bokoch GM, Balch WE. Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes. J Cell Biol. 1992 Dec;119(5):1077–1096. [PMC free article] [PubMed]
  • Shaw JM, Wickner WT. vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting. EMBO J. 1991 Jul;10(7):1741–1748. [PMC free article] [PubMed]
  • Stow JL, de Almeida JB, Narula N, Holtzman EJ, Ercolani L, Ausiello DA. A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells. J Cell Biol. 1991 Sep;114(6):1113–1124. [PMC free article] [PubMed]
  • Sukumar M, Higashijima T. G protein-bound conformation of mastoparan-X, a receptor-mimetic peptide. J Biol Chem. 1992 Oct 25;267(30):21421–21424. [PubMed]
  • Sullivan KM, Busa WB, Wilson KL. Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. Cell. 1993 Jul 2;73(7):1411–1422. [PubMed]
  • Tan A, Bolscher J, Feltkamp C, Ploegh H. Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S. J Cell Biol. 1992 Mar;116(6):1357–1367. [PMC free article] [PubMed]
  • Taylor TC, Kahn RA, Melançon P. Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-Golgi transport. Cell. 1992 Jul 10;70(1):69–79. [PubMed]
  • Tomita U, Inanobe A, Kobayashi I, Takahashi K, Ui M, Katada T. Direct interactions of mastoparan and compound 48/80 with GTP-binding proteins. J Biochem. 1991 Jan;109(1):184–189. [PubMed]
  • Tooze SA, Weiss U, Huttner WB. Requirement for GTP hydrolysis in the formation of secretory vesicles. Nature. 1990 Sep 13;347(6289):207–208. [PubMed]
  • Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS, Wittinghofer A. Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J. 1986 Jun;5(6):1351–1358. [PMC free article] [PubMed]
  • Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev. 1992 Sep;56(3):395–411. [PMC free article] [PubMed]
  • Vater CA, Raymond CK, Ekena K, Howald-Stevenson I, Stevens TH. The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains. J Cell Biol. 1992 Nov;119(4):773–786. [PMC free article] [PubMed]
  • Vitale N, Mukai H, Rouot B, Thiersé D, Aunis D, Bader MF. Exocytosis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein G(o). J Biol Chem. 1993 Jul 15;268(20):14715–14723. [PubMed]
  • Wallace MA, Carter HR. Effects of the wasp venom peptide, mastoparan, on a phosphoinositide-specific phospholipase C purified from rabbit brain membranes. Biochim Biophys Acta. 1989 Dec 18;1006(3):311–316. [PubMed]
  • Weingarten R, Ransnäs L, Mueller H, Sklar LA, Bokoch GM. Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi. J Biol Chem. 1990 Jul 5;265(19):11044–11049. [PubMed]
  • Weisman LS, Wickner W. Intervacuole exchange in the yeast zygote: a new pathway in organelle communication. Science. 1988 Jul 29;241(4865):589–591. [PubMed]
  • Weisman LS, Wickner W. Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting. J Biol Chem. 1992 Jan 5;267(1):618–623. [PubMed]
  • Weisman LS, Emr SD, Wickner WT. Mutants of Saccharomyces cerevisiae that block intervacuole vesicular traffic and vacuole division and segregation. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1076–1080. [PMC free article] [PubMed]
  • Wilson BS, Palade GE, Farquhar MG. Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1681–1685. [PMC free article] [PubMed]
  • Yamanaka G, Eckstein F, Stryer L. Interaction of retinal transducin with guanosine triphosphate analogues: specificity of the gamma-phosphate binding region. Biochemistry. 1986 Oct 7;25(20):6149–6153. [PubMed]
  • Yamashiro CT, Kane PM, Wolczyk DF, Preston RA, Stevens TH. Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase. Mol Cell Biol. 1990 Jul;10(7):3737–3749. [PMC free article] [PubMed]
  • Zeuzem S, Feick P, Zimmermann P, Haase W, Kahn RA, Schulz I. Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6619–6623. [PMC free article] [PubMed]

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