scholarly journals A Rab prenyl membrane-anchor allows effector recognition to be regulated by guanine nucleotide

2020 ◽  
Vol 117 (14) ◽  
pp. 7739-7744
Author(s):  
Miriam Lee ◽  
William Wickner ◽  
Hongki Song
Keyword(s):  
Membrane Fusion ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Membrane Anchor ◽  
Gtpase Activating Proteins ◽  
Fusion Inhibition ◽  
Additional Mode ◽  
Transmembrane Anchor ◽  
Effector Recognition ◽  
Hydrolysis Of

Membrane fusion is catalyzed by conserved proteins R, Qa, Qb, and Qc SNAREs, which form tetrameric RQaQbQc complexes between membranes; SNARE chaperones of the SM, Sec17/αSNAP, and Sec18/NSF families; Rab-GTPases (Rabs); and Rab effectors. Rabs are anchored to membranes by C-terminal prenyl groups, but can also function when anchored by an apolar polypeptide. Rabs are regulated by GTPase-activating proteins (GAPs), activating the hydrolysis of bound GTP. We have reconstituted fusion with pure components from yeast vacuoles including SNAREs, the HOPS (homotypic fusion and vacuole protein sorting) tethering and SNARE-assembly complex, and the Rab Ypt7, bound to membranes by either C-terminal prenyl groups (Ypt7-pr) or a recombinant transmembrane anchor (Ypt7-tm). We now report that HOPS-dependent fusion occurs with Ypt7 anchored by either means, but only Ypt7-pr requires GTP for activation and is inactive either with bound GDP or without bound guanine nucleotide. In contrast, Ypt7-tm is constitutively active for HOPS-dependent fusion, independent of bound guanine nucleotide. Fusion inhibition by the GAP Gyp1-46 is not limited to Ypt7-tm with bound GTP, indicating that this GAP has an additional mode of regulating fusion. Phosphorylation of HOPS by the vacuolar kinase Yck3 renders fusion strictly dependent on GTP-activated Ypt7, whether bound to membranes by prenyl or transmembrane anchor. The binding of GTP or GDP constitutes a selective switch for Ypt7, but with Ypt7-tm, this switch is only read by HOPS after phosphorylation to P-HOPS by its physiological kinase Yck3. The prenyl anchor of Ypt7 allows both HOPS and P-HOPS to be regulated by Ypt7-bound guanine nucleotide.

New chemical method of viral inactivation for vaccine development based on membrane fusion inhibition

Vaccine ◽  
2007 ◽  
Vol 25 (46) ◽  
pp. 7885-7892 ◽  
Author(s):  
Fausto Stauffer ◽  
Joari De Miranda ◽  
Marcos C. Schechter ◽  
Fernando A. Queiroz ◽  
Nathalia O. Santos ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Chemical Method ◽  
Vaccine Development ◽  
Viral Inactivation ◽  
Fusion Inhibition

scholarly journals RAP, a member of the Ras superfamily of small GTPases and its putative GTPase activating proteins and guanine nucleotide exchange factors in raw 264.7 macrophages

2021 ◽  
Author(s):  
Monika Tucholska
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Raw 264.7 ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Fcγ Receptor ◽  
Raw 264.7 Macrophages ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Ras Superfamily

The Fcγ receptor is a cell surface protein essential in the immune response that binds IgG-opsonized particles resulting in phagocytosis. Phagocytosis is a process used to remove pathogens and confine them in a vacuole that will enable their breakdown. The members of the Ras superfamily of small G proteins have been identified in samples where the activated Fcγ receptor complex was captured and analyzed using tandem mass spectrometry. The protein Rap. beloning to the Ras superfamily, guanosine triphosphatases (GTPase) activating proteins (GAPs), which promote the dissociation of GTP, and guanine nucleotide exchange factors (GEFs), that permits the exchange of GDP for GTP, were detected by SEQUEST in RAW 264.7 macrophages and futher analyzed using various methods. In this study, Raps, RasGAPs, and RapGEFs, were observed by tandem mass spectrometry and sequence correlation analysis. The selected isoforms were confirmed by Western blots, live cell confocal microscopy with fluorescent fusion constructs and antibody staining to verify the localization of Ras proetins, specifically Rap1, p120RasGAP and C3G, a RapGEF, to activated Fc reeceptor [sic].

scholarly journals Hormones Secretion and Rho GTPases in Neuroendocrine Tumors

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1859
Author(s):  
Laura Streit ◽  
Laurent Brunaud ◽  
Nicolas Vitale ◽  
Stéphane Ory ◽  
Stéphane Gasman
Keyword(s):  
Neuroendocrine Tumors ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Secretory Activity ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins

Neuroendocrine tumors (NETs) belong to a heterogeneous group of neoplasms arising from hormone secreting cells. These tumors are often associated with a dysfunction of their secretory activity. Neuroendocrine secretion occurs through calcium-regulated exocytosis, a process that is tightly controlled by Rho GTPases family members. In this review, we compiled the numerous mutations and modification of expression levels of Rho GTPases or their regulators (Rho guanine nucleotide-exchange factors and Rho GTPase-activating proteins) that have been identified in NETs. We discussed how they might regulate neuroendocrine secretion.

scholarly journals Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

2019 ◽  
Vol 116 (47) ◽  
pp. 23573-23581
Author(s):  
Youngsoo Jun ◽  
William Wickner
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Complex ◽  
Negative Regulation ◽  
Rab Gtpases ◽  
Sm Proteins ◽  
Rab Family ◽  
Sm Protein

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

scholarly journals The Effect of F-actin on the Binding and Hydrolysis of Guanine Nucleotide byDictyosteliumElongation Factor 1A

1998 ◽  
Vol 273 (17) ◽  
pp. 10288-10295 ◽  
Author(s):  
Brian T. Edmonds ◽  
Andrea Bell ◽  
Jeffrey Wyckoff ◽  
John Condeelis ◽  
Thomas S. Leyh
Keyword(s):  
Guanine Nucleotide ◽  
Hydrolysis Of

Control of Exocytosis in Single Cells

Physiology ◽  
1989 ◽  
Vol 4 (2) ◽  
pp. 53-56 ◽  
Author(s):  
Y Maruyama
Keyword(s):  
Membrane Fusion ◽  
G Proteins ◽  
Secretory Granule ◽  
Single Cells ◽  
Guanine Nucleotide ◽  
Signal Transducers ◽  
Granule Membrane ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Exocytosis can be quantified by measuring changes in membrane capacitance in single internally perfused cells. Exocytosis is controlled by guanine nucleotide-binding proteins (G proteins) acting as key signal transducers. Different G proteins mediate receptor signaling and secretory granule-membrane fusion.

scholarly journals Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes

1985 ◽  
Vol 232 (3) ◽  
pp. 799-804 ◽  
Author(s):  
R A Gonzales ◽  
F T Crews
Keyword(s):  
Inositol Phosphates ◽  
Adenine Nucleotides ◽  
Inositol Trisphosphate ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Inositol Phospholipids ◽  
Cortical Membranes ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Stimulation Of

The guanine nucleotides guanosine 5′[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5′-[γ-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H)inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.

scholarly journals GTPase-Activating Proteins for Cdc42

2002 ◽  
Vol 1 (3) ◽  
pp. 469-480 ◽  
Author(s):  
Gregory R. Smith ◽  
Scott A. Givan ◽  
Paul Cullen ◽  
George F. Sprague
Keyword(s):  
Rho Gtpase ◽  
Invasive Growth ◽  
Gtpase Activating Proteins ◽  
Morphological Defects ◽  
P21 Activated Kinase ◽  
The Individual ◽  
Hydrolysis Of ◽  
Pak Kinase

ABSTRACT The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of RGA1, but not RGA2 or BEM3, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of BEM3 resulted in cells with severe morphological defects not observed in rga1Δ or rga2Δ strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.

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