scholarly journals Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole

2012 ◽  
Vol 23 (7) ◽  
pp. 1267-1282 ◽  
Author(s):  
Miriam Essid ◽  
Navin Gopaldass ◽  
Kunito Yoshida ◽  
Christien Merrifield ◽  
Thierry Soldati
Keyword(s):  
Plasma Membrane ◽  
Dominant Negative ◽  
Contractile Vacuole ◽  
Dominant Negative Mutant ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Exocyst Complex ◽  
Nucleotide Exchange Factor ◽  
Constitutively Active

Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak–Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.

scholarly journals The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane

2005 ◽  
Vol 281 (5) ◽  
pp. 2506-2514 ◽  
Author(s):  
Yu Li ◽  
Sirisha Asuri ◽  
John F. Rebhun ◽  
Ariel F. Castro ◽  
Nivanka C. Paranavitana ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

scholarly journals Active Arf6 Recruits ARNO/Cytohesin GEFs to the PM by Binding Their PH Domains

2007 ◽  
Vol 18 (6) ◽  
pp. 2244-2253 ◽  
Author(s):  
Lee Ann Cohen ◽  
Akira Honda ◽  
Peter Varnai ◽  
Fraser D. Brown ◽  
Tamas Balla ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Family Member ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Biochemical Assays ◽  
Inositol Phospholipids ◽  
Sec7 Domain

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

scholarly journals Identification of a Plasma Membrane-associated Guanine Nucleotide Exchange Factor for ARF6 in Chromaffin Cells

2000 ◽  
Vol 275 (21) ◽  
pp. 15637-15644 ◽  
Author(s):  
Anne-Sophie Caumont ◽  
Nicolas Vitale ◽  
Marc Gensse ◽  
Marie-Christine Galas ◽  
James E. Casanova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

scholarly journals Regulation of type V adenylate cyclase by Ric8a, a guanine nucleotide exchange factor

2007 ◽  
Vol 406 (3) ◽  
pp. 383-388 ◽  
Author(s):  
Shyi-Chyi Wang ◽  
Hsing-Lin Lai ◽  
Yi-Ting Chiu ◽  
Ren Ou ◽  
Chuen-Lin Huang ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Guanine Nucleotide Exchange Factor ◽  
Dominant Negative ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
N Terminus ◽  
Type V

In the present study, we demonstrate that AC5 (type V adenylate cyclase) interacts with Ric8a through directly interacting at its N-terminus. Ric8a was shown to be a GEF (guanine nucleotide exchange factor) for several α subunits of heterotrimeric GTP binding proteins (Gα proteins) in vitro. Selective Gα targets of Ric8a have not yet been revealed in vivo. An interaction between AC5 and Ric8a was verified by pull-down assays, co-immunoprecipitation analyses, and co-localization in the brain. Expression of Ric8a selectively suppressed AC5 activity. Treating cells with pertussis toxin or expressing a dominant negative Gαi mutant abolished the suppressive effect of Ric8a, suggesting that interaction between the N-terminus of AC5 and a GEF (Ric8a) provides a novel pathway to fine-tune AC5 activity via a Gαi-mediated pathway.

scholarly journals Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1

2004 ◽  
Vol 382 (3) ◽  
pp. 857-865 ◽  
Author(s):  
Ian N. FLEMING ◽  
Ian H. BATTY ◽  
Alan R. PRESCOTT ◽  
Alex GRAY ◽  
Gursant S. KULAR ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Inositol Phosphates ◽  
Pleckstrin Homology Domain ◽  
Membrane Association ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Pleckstrin Homology ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P2 to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P2 breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P3 concentrations, rather than the closely related lipid, PtdIns(3,4)P2. Finally, the data demonstrate that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.

A Role for the Noncatalytic N Terminus in the Function of Cdc25, a Saccharomyces cerevisiae Ras-Guanine Nucleotide Exchange Factor

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1473-1484
Author(s):  
Reneé A Chen ◽  
Tamer Michaeli ◽  
Linda Van Aelst ◽  
Roymarie Ballester
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Guanine Nucleotide Exchange Factor ◽  
Dominant Negative ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ras Pathway ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
N Terminus

Abstract The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor (GEF) for Ras proteins. Its catalytic domain is highly homologous to Ras-GEFs from all eukaryotes. Even though Cdc25 is the first Ras-GEF identified in any organism, we still know very little about how its function is regulated in yeast. In this work we provide evidence for the involvement of the N terminus of Cdc25 in the regulation of its activity. A truncated CDC25 lacking the noncatalytic C-terminal coding sequence was identified in a screen of high-copy suppressors of the heat-shock-sensitive phenotype of strains in which the Ras pathway is hyper-activated. The truncated gene acts as a dominant-negative mutant because it only suppresses the heat-shock sensitivity of strains that require the function of CDC25. Our two-hybrid assays and immunoprecipitation analyses show interactions between the N terminus of Cdc25 and itself, the C terminus, and the full-length protein. These results suggest that the dominant-negative effect may be a result of oligomerization with endogenous Cdc25. Further evidence of the role of the N terminus of Cdc25 in the regulation of its activity is provided by the mapping of the activating mutation of CDC25HS20 to the serine residue at position 365 in the noncatalytic N-terminal domain. This mutation induces a phenotype similar to activating mutants of other genes in the Ras pathway in yeast. Hence, the N terminus may exert a negative control on the catalytic activity of the protein. Taken together these results suggest that the N terminus plays a crucial role in regulating Cdc25 and consequently Ras activity, which in S. cerevisiae is essential for cell cycle progression.

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