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.

scholarly journals Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER.

1991 ◽  
Vol 114 (4) ◽  
pp. 663-670 ◽  
Author(s):  
C d'Enfert ◽  
L J Wuestehube ◽  
T Lila ◽  
R Schekman
Keyword(s):  
Membrane Protein ◽  
Protein Transport ◽  
Binding Protein ◽  
Integral Membrane Protein ◽  
Gtp Binding Protein ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Transport Vesicles

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.

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 The small GTP-binding protein rab6 functions in intra-Golgi transport.

1994 ◽  
Vol 127 (6) ◽  
pp. 1575-1588 ◽  
Author(s):  
O Martinez ◽  
A Schmidt ◽  
J Salaméro ◽  
B Hoflack ◽  
M Roa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Wild Type ◽  
Gtp Binding Protein ◽  
Anterograde Transport ◽  
Golgi Transport ◽  
Gtp Binding ◽  
Hemagglutinin Protein

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Géraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.

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 The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

1989 ◽  
Vol 109 (3) ◽  
pp. 1015-1022 ◽  
Author(s):  
R A Bacon ◽  
A Salminen ◽  
H Ruohola ◽  
P Novick ◽  
S Ferro-Novick
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Transport Defect

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

Overexpression of developmentally regulated GTP-binding protein-2 increases bone loss

2013 ◽  
Vol 304 (7) ◽  
pp. E703-E710 ◽  
Author(s):  
Ke Ke ◽  
Ok-Joo Sul ◽  
Woon-Ki Kim ◽  
Mi-Hyun Lee ◽  
Myung-Seok Ko ◽  
...  
Keyword(s):  
Bone Loss ◽  
Bone Mass ◽  
Binding Protein ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Developmentally Regulated ◽  
Gtp Binding ◽  
Actin Rings

The developmentally regulated GTP-binding protein-2 (DRG2) is a novel subclass of GTP-binding proteins. Many functional characteristics of osteoclasts (OC) are associated with small GTPases. We hypothesized that DRG2 affects bone mass via modulating OC activity. Using DRG2 transgenic mice, we investigated the role of DRG2 in bone remodeling. DRG2 overexpression caused a decrease in bone mass and an increase in the number and activity of OC in vivo. DRG2 overexpression increased fusion, spreading, survival, and resorption activity of OC in vitro . Downregulation of DRG2 by siRNA decreased fusion, spreading, and survival of OC, supporting the observations found in DRG2 transgenic OC. Transgenic mature OCs were larger, with actin rings and higher ERK, Akt, Rac1 and Rho activities than wild-type OCs. Inhibition of these proteins abolished the effects of DRG2 on formation of large OCs with actin rings, implying that DRG2 affects cytoskeleton reorganization in a Rac1/Rho/ERK/Akt-dependent manner. In summary, DRG2 is associated with survival and cytoskeleton organization of OC under influence of macrophage colony-stimulating factor, and its overexpression leads to elevated bone resorptive activity of OC, resulting in bone loss.

scholarly journals Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

2015 ◽  
Vol 112 (28) ◽  
pp. E3679-E3688 ◽  
Author(s):  
Susanne de Boor ◽  
Philipp Knyphausen ◽  
Nora Kuhlmann ◽  
Sarah Wroblowski ◽  
Julian Brenig ◽  
...  
Keyword(s):  
Binding Protein ◽  
Rat Tissues ◽  
Lysine Acetylation ◽  
Nucleotide Exchange ◽  
Gtp Binding Protein ◽  
Cellular Effects ◽  
Gtp Binding ◽  
Cytoplasmic Transport

Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.

Light-modulated subcellular localization of the alpha-subunit of GTP-binding protein Gq in crayfish photoreceptors

1996 ◽  
Vol 13 (3) ◽  
pp. 539-547 ◽  
Author(s):  
Akihisa Terakita ◽  
Hideki Takahama ◽  
Satoshi Tamotsu ◽  
Tatsuo Suzuki ◽  
Takahiko Hariyama ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Binding Protein ◽  
Light Condition ◽  
Alpha Subunit ◽  
Soluble Form ◽  
Acid Modification ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Membrane Bound

AbstractGq-type GTP-binding protein (Gq) plays an important role in invertebrate visual phototransduction. The subcellular localization of the alpha subunit of visual Gq in crayfish photoreceptor was investigated immunocytochemically and biochemically to demonstrate the details of the rhodopsin-Gq interaction. The localization of Gq(alpha) changed depending on the light condition. In the dark, Gq(alpha) was localized in the whole rhabdoms as the membrane-bound form. In the light, half of the Gq(alpha) was localized in the cytoplasm as the soluble form. The translocation of Gq(alpha) was reversible. The light-modulated translocation possibly controls the amount of Gq that can be activated by rhodopsin. In vitro hydroxylamine treatment of rhabdomeric membranes suggested that the translocation was regulated by the fatty-acid modification of Gq(alpha).

scholarly journals Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein.

1986 ◽  
Vol 103 (6) ◽  
pp. 2253-2261 ◽  
Author(s):  
T Connolly ◽  
R Gilmore
Keyword(s):  
Signal Sequence ◽  
Binding Protein ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Signal Recognition ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Protease Digestion ◽  
Nascent Chain

The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt-insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide-independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.

The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro

Neuron ◽  
1990 ◽  
Vol 4 (5) ◽  
pp. 797-805 ◽  
Author(s):  
J. Ayala ◽  
N. Touchot ◽  
A. Zahraoui ◽  
A. Tavitian ◽  
A. Prochiantz
Keyword(s):  
Binding Protein ◽  
Neurite Growth ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Neuronal Adhesion
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