Home LiteratureArticle Details
PMID: 11598009 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Cdc42p functions at the docking stage of yeast vacuole membrane fusion.

The EMBO journal ·Vol. 20 ·No. 20 ·2001-10-15 ·Pages 5657-65

Müller O, Johnson DI, Mayer A

Abstract

Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases. In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a member of the Rho family of GTPases, has a direct role in membrane fusion. Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery. Vacuoles from cdc42 temperature-sensitive mutants are deficient for fusion at the restrictive temperature. Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion. Affinity-purified antibodies to this domain inhibited the in vitro fusion reaction. Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post-docking phase of the reaction, we show that Cdc42p action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes. Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.

MeSH Terms
Adenosine Triphosphatases Alleles Calcium/metabolism Carrier Proteins/metabolism Chelating Agents/pharmacology Egtazic Acid/analogs & derivatives,pharmacology Fungal Proteins/chemistry,genetics,metabolism,physiology Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology Macromolecular Substances Membrane Fusion Membrane Proteins/metabolism Molecular Chaperones Protein Transport Recombinant Fusion Proteins/physiology SNARE Proteins Saccharomyces cerevisiae/genetics,metabolism,ultrastructure Saccharomyces cerevisiae Proteins Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins Vacuoles/physiology Vesicular Transport Proteins cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/chemistry,genetics,physiology rab GTP-Binding Proteins/genetics,physiology
Chemicals
Carrier Proteins Chelating Agents Fungal Proteins Macromolecular Substances Membrane Proteins Molecular Chaperones Recombinant Fusion Proteins SEC17 protein, S cerevisiae SNARE Proteins Saccharomyces cerevisiae Proteins Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins VTC1 protein, S cerevisiae Vesicular Transport Proteins Guanosine 5'-O-(3-Thiotriphosphate) Egtazic Acid Adenosine Triphosphatases SEC18 protein, S cerevisiae YPT7 protein, S cerevisiae cdc42 GTP-Binding Protein, Saccharomyces cerevisiae rab GTP-Binding Proteins 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid Calcium
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Müller O
Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, 72076 Tübingen, Germany.
Johnson D I
Mayer A
References (88)
88 references, click to expand
  1. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.
    Nature. 1998 Sep 24;395(6700):347-53 PMID: 9759724
  2. Identification of the binding surface on Cdc42Hs for p21-activated kinase.
    Biochemistry. 1998 Oct 6;37(40):14030-7 PMID: 9760238
  3. Defining the functions of trans-SNARE pairs.
    Nature. 1998 Dec 10;396(6711):543-8 PMID: 9859990
  4. Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion.
    Nature. 1998 Dec 10;396(6711):575-80 PMID: 9859992
  5. The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42.
    Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15362-7 PMID: 9860974
  6. Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity.
    Microbiol Mol Biol Rev. 1999 Mar;63(1):54-105 PMID: 10066831
  7. Activation of the Cdc42-associated tyrosine kinase-2 (ACK-2) by cell adhesion via integrin beta1.
    J Biol Chem. 1999 Mar 26;274(13):8524-30 PMID: 10085085
  8. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly.
    Cell. 1999 Apr 16;97(2):221-31 PMID: 10219243
  9. Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.
    Nature. 1999 May 27;399(6734):379-83 PMID: 10360578
  10. Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK.
    Nature. 1999 May 27;399(6734):384-8 PMID: 10360579
  11. The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast.
    J Biol Chem. 1999 Jun 11;274(24):16861-70 PMID: 10358031
  12. Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion.
    J Cell Biol. 1999 Jun 28;145(7):1435-42 PMID: 10385523
  13. Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.
    Science. 1999 Aug 13;285(5430):1084-7 PMID: 10446058
  14. Membrane tethering in intracellular transport.
    Curr Opin Cell Biol. 1999 Aug;11(4):453-9 PMID: 10449330
  15. Rab proteins as membrane organizers.
    Nat Rev Mol Cell Biol. 2001 Feb;2(2):107-17 PMID: 11252952
  16. Spatial regulation of the exocyst complex by Rho1 GTPase.
    Nat Cell Biol. 2001 Apr;3(4):353-60 PMID: 11283608
  17. The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe.
    J Biol Chem. 2001 Feb 2;276(5):3004-9 PMID: 11042180
  18. Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion.
    EMBO J. 2001 Jun 15;20(12):3145-55 PMID: 11406591
  19. Fusion of docked membranes requires the armadillo repeat protein Vac8p.
    J Biol Chem. 2001 Sep 14;276(37):35133-40 PMID: 11441010
  20. Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking.
    EMBO J. 2001 Oct 15;20(20):5650-6 PMID: 11598008
  21. Pancreatic beta-cell web: its possible role in insulin secretion.
    Science. 1972 Mar 10;175(4026):1128-30 PMID: 4551150
  22. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.
    Anal Biochem. 1984 Apr;138(1):141-3 PMID: 6731838
  23. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.
    J Cell Biol. 1990 Jul;111(1):131-42 PMID: 2195038
  24. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity.
    J Cell Biol. 1990 Jul;111(1):143-52 PMID: 2164028
  25. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.
    Biophys J. 2000 Jun;78(6):2863-77 PMID: 10827968
  26. The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation.
    Nature. 2000 Jun 15;405(6788):800-4 PMID: 10866202
  27. GTP hydrolysis by Ran is required for nuclear envelope assembly.
    Mol Cell. 2000 Jun;5(6):1013-24 PMID: 10911995
  28. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.
    Cell. 1999 Aug 6;98(3):377-86 PMID: 10458612
  29. ATP-dependent membrane assembly of F-actin facilitates membrane fusion.
    Mol Biol Cell. 2001 Jan;12(1):155-70 PMID: 11160830
  30. Multiple roles for Cdc42 in cell regulation.
    Curr Opin Cell Biol. 2001 Apr;13(2):153-7 PMID: 11248548
  31. PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae.
    J Cell Biol. 1999 Nov 15;147(4):845-56 PMID: 10562285
  32. Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells.
    Nat Cell Biol. 1999 May;1(1):8-13 PMID: 10559857
  33. The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity.
    Mol Biol Cell. 1999 Dec;10(12):4121-33 PMID: 10588647
  34. Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe.
    Genetics. 2000 Jan;154(1):155-65 PMID: 10628977
  35. Functions and functional domains of the GTPase Cdc42p.
    Mol Biol Cell. 2000 Jan;11(1):339-54 PMID: 10637312
  36. The cellular biology of proton-motive force generation by V-ATPases.
    J Exp Biol. 2000 Jan;203(Pt 1):89-95 PMID: 10600677
  37. Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells.
    J Cell Biol. 2000 Feb 7;148(3):481-94 PMID: 10662774
  38. Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion.
    Mol Biol Cell. 2000 Mar;11(3):807-17 PMID: 10712501
  39. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein.
    Nature. 2000 Mar 9;404(6774):151-8 PMID: 10724160
  40. Proteins needed for vesicle budding from the Golgi complex are also required for the docking step of homotypic vacuole fusion.
    J Cell Biol. 2000 Mar 20;148(6):1223-29 PMID: 10725335
  41. The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein.
    J Cell Biol. 2000 Mar 20;148(6):1231-8 PMID: 10725336
  42. A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion.
    Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9402-7 PMID: 10944212
  43. Developmental control of endocytosis in dendritic cells by Cdc42.
    Cell. 2000 Aug 4;102(3):325-34 PMID: 10975523
  44. Yeast homotypic vacuole fusion: a window on organelle trafficking mechanisms.
    Annu Rev Biochem. 2000;69:247-75 PMID: 10966459
  45. Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.
    J Cell Biol. 2000 Sep 18;150(6):1299-310 PMID: 10995436
  46. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex.
    J Cell Biol. 2000 Sep 18;150(6):1311-20 PMID: 10995437
  47. Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion.
    Mol Cell. 2000 Sep;6(3):661-71 PMID: 11030345
  48. Small G-protein networks: their crosstalk and signal cascades.
    Cell Signal. 2000 Aug;12(8):515-24 PMID: 11027944
  49. Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex.
    Science. 2000 Oct 27;290(5492):801-6 PMID: 11052943
  50. New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion.
    J Cell Biol. 2000 Oct 30;151(3):551-62 PMID: 11062257
  51. Sequential action of two GTPases to promote vacuole docking and fusion.
    EMBO J. 2000 Dec 15;19(24):6713-20 PMID: 11118206
  52. Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton.
    Curr Biol. 2000 Dec 14-28;10(24):1587-90 PMID: 11137010
  53. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion.
    Nature. 2001 Feb 1;409(6820):581-8 PMID: 11214310
  54. Atomic model of the actin filament.
    Nature. 1990 Sep 6;347(6288):44-9 PMID: 2395461
  55. Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily.
    Mol Cell Biol. 1991 Feb;11(2):872-85 PMID: 1990290
  56. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
    Anal Biochem. 1991 Feb 1;192(2):262-7 PMID: 1852137
  57. Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.
    Mol Cell Biol. 1991 Jul;11(7):3537-44 PMID: 1904541
  58. A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42.
    Nature. 1993 May 27;363(6427):364-7 PMID: 8497321
  59. Cytoskeleton dynamics during neurotransmitter release.
    Trends Neurosci. 1993 Nov;16(11):466-72 PMID: 7507620
  60. Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.
    Mol Biol Cell. 1993 Oct;4(10):1017-25 PMID: 8298188
  61. Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.
    Mol Biol Cell. 1993 Dec;4(12):1307-16 PMID: 8167411
  62. Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro.
    J Cell Biol. 1994 Jul;126(1):99-110 PMID: 8027190
  63. Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis.
    Neuron. 1995 Feb;14(2):353-63 PMID: 7857644
  64. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells.
    J Cell Biol. 1995 Feb;128(4):589-98 PMID: 7860632
  65. Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast.
    J Cell Biol. 1995 Feb;128(4):599-615 PMID: 7860633
  66. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.
    Mol Cell Biol. 1995 Apr;15(4):1942-52 PMID: 7891688
  67. The role of Myo2, a yeast class V myosin, in vesicular transport.
    J Cell Biol. 1995 Mar;128(6):1055-68 PMID: 7896871
  68. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
    Cell. 1995 Apr 7;81(1):53-62 PMID: 7536630
  69. Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast.
    Genes Dev. 1995 Aug 1;9(15):1817-30 PMID: 7649470
  70. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases.
    J Biol Chem. 1995 Dec 8;270(49):29071-4 PMID: 7493928
  71. Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles.
    Cell. 1996 Apr 5;85(1):83-94 PMID: 8620540
  72. A new efficient gene disruption cassette for repeated use in budding yeast.
    Nucleic Acids Res. 1996 Jul 1;24(13):2519-24 PMID: 8692690
  73. Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus.
    J Biol Chem. 1996 Oct 25;271(43):26850-4 PMID: 8900167
  74. Endosome dynamics regulated by a Rho protein.
    Nature. 1996 Dec 5;384(6608):427-32 PMID: 8945468
  75. Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae.
    J Cell Biol. 1996 Dec;135(6 Pt 1):1535-49 PMID: 8978821
  76. Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF).
    J Cell Biol. 1997 Jan 27;136(2):307-17 PMID: 9015302
  77. Homotypic vacuolar fusion mediated by t- and v-SNAREs.
    Nature. 1997 May 8;387(6629):199-202 PMID: 9144293
  78. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.
    J Biol Chem. 1997 Aug 15;272(33):20332-5 PMID: 9252336
  79. Characterization of synthetic-lethal mutants reveals a role for the Saccharomyces cerevisiae guanine-nucleotide exchange factor Cdc24p in vacuole function and Na+ tolerance.
    Genetics. 1997 Sep;147(1):43-55 PMID: 9286667
  80. Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain.
    J Biol Chem. 1997 Oct 3;272(40):24819-24 PMID: 9312079
  81. A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion.
    J Cell Biol. 1998 Jan 12;140(1):61-9 PMID: 9425154
  82. Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell?
    Trends Biochem Sci. 1997 Dec;22(12):468-72 PMID: 9433126
  83. Actin disassembles reversibly during electrically induced recycling of synaptic vesicles in cultured neurons.
    Brain Res Mol Brain Res. 1998 Jan;53(1-2):236-51 PMID: 9473683
  84. Vac8p, a vacuolar protein with armadillo repeats, functions in both vacuole inheritance and protein targeting from the cytoplasm to vacuole.
    J Cell Biol. 1998 Mar 9;140(5):1063-74 PMID: 9490720
  85. Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts.
    J Cell Biol. 1998 Mar 9;140(5):1125-36 PMID: 9490725
  86. Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.
    EMBO J. 1998 Apr 15;17(8):2156-65 PMID: 9545229
  87. Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42.
    Mol Biol Cell. 1998 May;9(5):1053-63 PMID: 9571239
  88. LMA1 binds to vacuoles at Sec18p (NSF), transfers upon ATP hydrolysis to a t-SNARE (Vam3p) complex, and is released during fusion.
    Cell. 1998 Jun 26;93(7):1125-34 PMID: 9657146
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/20.20.5657
Published
2001-10-15
Pages
5657-65
Language
English
Region
England
NLM ID
8208664
PMCID
PMC125663
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/20.20.5657 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2