scholarly journals Fusion between rat pancreatic zymogen granules and plasma membranes. Modulation by a GTP-binding protein

1992 ◽  
Vol 286 (3) ◽  
pp. 747-753 ◽  
Author(s):  
C M MacLean ◽  
J M Edwardson
Keyword(s):  
Plasma Membranes ◽  
Binding Protein ◽  
Molecular Characteristics ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Fusion Event ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Wide Range

At the moment, little is known about the molecular characteristics of the final step in the process of regulated exocytosis, i.e. the fusion of the membrane of a secretory vesicle with the plasma membrane. We have reconstituted this fusion event in vitro, using zymogen granules and plasma membranes from the exocrine pancreas of the rat. The membranes of zymogen granules were loaded with the lipid-soluble fluorescent probe octadecylrhodamine B, at a concentration that resulted in self-quenching of its fluorescence. The granules were then incubated with pancreatic plasma membranes at 37 degrees C, and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Zymogen granules fused with pancreatic plasma membranes, but not with plasma membranes from liver or chromaffin cells; granules also fused with unlabelled granule membranes. The fusion of granules with plasma membranes was unaffected by variation of the Ca2+ concentration over a wide range, but fusion of granules with both plasma membranes and zymogen granule membranes was stimulated by GTP and, more potently, by guanosine 5′-[gamma-thio]triphosphate (GTP[S]). The effect of GTP[S] was to increase the extent of fusion occurring at low concentrations of plasma membranes, without affecting the maximum signal obtained at high membrane concentrations. Pre-incubation of the plasma membranes with GTP[S] also enhanced their ability to fuse with zymogen granules. Our results indicate that membrane fusion during exocytosis may be under the direct control of a GTP-binding protein.

scholarly journals Reconstitution in vitro of a membrane-fusion event involved in constitutive exocytosis. A role for cytosolic proteins and a GTP-binding protein, but not for Ca2+

1992 ◽  
Vol 285 (2) ◽  
pp. 383-385 ◽  
Author(s):  
J M Edwardson ◽  
P U Daniels-Holgate
Keyword(s):  
Membrane Fusion ◽  
Binding Protein ◽  
Fusion Event ◽  
Gtp Binding Protein ◽  
In Vitro System ◽  
Cytosolic Proteins ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Transport Vesicles

The fusion of post-Golgi transport vesicles with the plasma membrane is perhaps the least well understood step in the network of intracellular membrane traffic. We have used an ‘in vitro’ system to study this membrane-fusion event. We show here that fusion requires the presence of cytosolic proteins, but not Ca2+, and is inhibited by the non-hydrolysable GTP analogue guanosine 5′-[gamma-thio]triphosphate, which indicates the involvement of a GTP-binding protein.

Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment

1986 ◽  
Vol 64 (4) ◽  
pp. 304-308 ◽  
Author(s):  
B. D. Gupta ◽  
T. J. Borys ◽  
S. Deshpande ◽  
R. E. Jones ◽  
E. W. Abrahamson
Keyword(s):  
Light Scattering ◽  
Outer Segment ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Rod Outer Segments ◽  
Particle Scattering ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Time Period ◽  
Hydrolysis Of

In the presence of exogeneous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition. GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5′-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.

scholarly journals Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics

1996 ◽  
Vol 316 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Stefan J. MARCINIAK ◽  
J. Michael EDWARDSON
Keyword(s):  
Plasma Membranes ◽  
Direct Action ◽  
Nucleoside Diphosphate Kinase ◽  
Pancreatic Acinar Cell ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Cytoplasmic Face ◽  
Granule Membrane ◽  
Stimulation Of

It is well established that both GTP-binding proteins and phosphoproteins are involved in the control of exocytosis in the exocrine pancreas. Exocytotic membrane fusion is stimulated by guanosine 5′-[γ-thio]triphosphate, and the phosphorylation states of several proteins, including at least one on the zymogen granule membrane, are known to change during exocytosis. We show here that a nucleoside diphosphate kinase is associated with the cytoplasmic face of pancreatic zymogen granules. This enzyme behaves as a phosphoprotein of apparent molecular mass 21 kDa on SDS/polyacrylamide gels, and is able to produce GTP by using ATP to phosphorylate endogenous GDP. GTP production by nucleoside diphosphate kinase is stimulated by the wasp venom peptide mastoparan, both through a direct action on the enzyme and through its ability to increase the availability of endogenous GDP. Two effects of the GTP produced by nucleoside diphosphate kinase are demonstrated: phosphorylation of a 37 kDa zymogen granule protein on histidine residues, and stimulation of the fusion of zymogen granules with pancreatic plasma membranes in vitro. These results suggest that granule-associated nucleoside diphosphate kinase is able to maintain local GTP concentrations, and raise the possibility that it might be involved in the control of exocytosis in the pancreatic acinar cell.

Localization of ral, a small Mr GTP-binding protein, to collecting duct cells in bovine and rat kidney

1991 ◽  
Vol 261 (6) ◽  
pp. F1063-F1070
Author(s):  
A. Gupta ◽  
B. Bastani ◽  
P. Chardin ◽  
K. A. Hruska
Keyword(s):  
Gene Product ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Gtp Binding Protein ◽  
Bovine Kidney ◽  
Gtp Binding ◽  
Collecting Ducts

Plasma membranes from bovine kidney cortex were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blotting with [alpha-32P]GTP and [35S]GTP gamma S demonstrated specific binding to three and six distinct protein bands, respectively, in the 20,000- to 29,000-Mr range. This indicated the presence of small Mr GTP binding proteins (smg) in bovine kidney cortex. Only one smg with 28,000 Mr was labeled with hydrolysis-resistant GTP photoaffinity probe p3-(4-azidoanilido)-p1-5GTP (AAGTP). The major smg in platelet membranes that binds GTP on nitrocellulose blots has been identified as ral-Mr 29,000. With the use of an antiserum against the ral A gene product, one of the smg with Mr of 29,000 present in bovine renal cortical plasma membranes was identified as ral. Ral was absent from glomerular homogenate, suggesting that it is localized to the tubular segments of the nephron. Ral was detected only in the particulate fraction and not the cytosol. Further subcellular localization of ral was investigated by immunohistochemical staining. Anti-ral antibody immunostained the apical and basolateral membranes of cells in the cortical and medullary collecting ducts in a speckled pattern in the bovine kidney. In the rat kidney, however, uniform linear staining of cortical and medullary collecting ducts predominantly localized to the apical membrane was observed. To date, no function has been assigned to ral. Localization of the ral gene product to the collecting duct suggests a specific functional role for this GTP-binding protein.

scholarly journals Purification and subunit composition of a GTP-binding protein from maize root plasma membranes

FEBS Letters ◽  
1991 ◽  
Vol 291 (2) ◽  
pp. 219-221 ◽  
Author(s):  
Sevo V. Bilushi ◽  
Alexander G. Shebunin ◽  
Alexey V. Babakov
Keyword(s):  
Plasma Membranes ◽  
Binding Protein ◽  
Maize Root ◽  
Subunit Composition ◽  
Gtp Binding Protein ◽  
Gtp Binding

scholarly journals Arf6 is necessary for high level Wingless signalling duringDrosophilawing development

2021 ◽  
Author(s):  
Julien Marcetteau ◽  
Tamàs Matusek ◽  
Frédéric Luton ◽  
Pascal P. Thérond
Keyword(s):  
Signal Transduction ◽  
Adherens Junctions ◽  
Developmental Model ◽  
Wing Margin ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Wide Range ◽  
High Level

AbstractWnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa.In vitrostudies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findingsin vivo, we analyse the consequence of the absence of Arf6 activity onDrosophilawing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.

scholarly journals A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas.

1991 ◽  
Vol 88 (20) ◽  
pp. 8925-8929 ◽  
Author(s):  
K. M. Warpeha ◽  
H. E. Hamm ◽  
M. M. Rasenick ◽  
L. S. Kaufman
Keyword(s):  
Blue Light ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding

scholarly journals GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion.

1989 ◽  
Vol 1 (1) ◽  
pp. 113-124 ◽  
Author(s):  
L S Mayorga ◽  
R Diaz ◽  
M I Colombo ◽  
P D Stahl
Keyword(s):  
Binding Protein ◽  
Limiting Factors ◽  
Gtp Binding Protein ◽  
Endosomal Membrane ◽  
Gtp Binding ◽  
High Concentrations ◽  
Endocytic Vesicles ◽  
Gamma S ◽  
Cytosolic Factor

Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.

scholarly journals Stimulation of exocytotic membrane fusion by modified peptides of the rab3 effector domain: re-evaluation of the role of rab3 in regulated exocytosis

1993 ◽  
Vol 294 (2) ◽  
pp. 325-328 ◽  
Author(s):  
C M MacLean ◽  
G J Law ◽  
J M Edwardson
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Fusion Event ◽  
Effector Domain ◽  
Granule Membrane ◽  
Modified Peptides ◽  
Stimulation Of

We have shown previously that fusion between pancreatic zymogen granules and plasma membranes is stimulated by a peptide corresponding to the putative effector domain of rab3. Here we show that this stimulatory effect persists when the amino acid sequence of the peptide is substantially modified. We also show that an antibody raised against rab3a recognizes a protein of appropriate size on the zymogen-granule membrane, but has no effect on membrane fusion. We suggest that rab3 is not directly involved in the control of this membrane fusion event, and that the peptides are stimulating fusion by a mechanism unrelated to rab3.

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