scholarly journals Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S.

1992 ◽  
Vol 116 (6) ◽  
pp. 1357-1367 ◽  
Author(s):  
A Tan ◽  
J Bolscher ◽  
C Feltkamp ◽  
H Ploegh
Keyword(s):  
Hepg2 Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Streptolysin O ◽  
Gamma S

The involvement of GTP-binding proteins in the intracellular transport of the secretory glycoprotein alpha 1-antitrypsin was investigated in streptolysin O-permeabilized HepG2 cells. This permeabilization procedure allows ready access to the intracellular milieu of the membrane-impermeant, nonhydrolyzable GTP analog GTP gamma S. In streptolysin O-permeabilized HepG2 cells, the constitutive secretory pathway remains functional and is sensitive to GTP gamma S. Exposure of HepG2 cells to brefeldin A resulted in redistribution of Golgi-resident glycosyltransferases (including both alpha 2----3 and alpha 2----6 sialyltransferases) to the ER. This redistribution was sensitive to GTP gamma S. Our results suggest that GTP-binding proteins are involved in the regulation not only of the anterograde, but also of the retrograde, pathway.

scholarly journals GTP gamma S inhibits organelle transport along axonal microtubules.

1993 ◽  
Vol 120 (2) ◽  
pp. 467-476 ◽  
Author(s):  
G S Bloom ◽  
B W Richards ◽  
P L Leopold ◽  
D M Ritchey ◽  
S T Brady
Keyword(s):  
Protein Kinases ◽  
Axonal Transport ◽  
Binding Proteins ◽  
Organelle Transport ◽  
Nucleotide Exchange ◽  
Fast Axonal Transport ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Organelle Motility ◽  
Gamma S

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubule-based organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTP gamma S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP beta S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP gamma S. The effects of GTP gamma S on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATP gamma S. Therefore, suppression of organelle motility by GTP gamma S was guanine nucleotide-specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by AlF4-, indicating that the GTP gamma S-sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP gamma S include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.

Role of G proteins in the acetylcholine-induced potassium current of canine atrial cells

1989 ◽  
Vol 257 (5) ◽  
pp. H1516-H1522
Author(s):  
S. Sorota ◽  
B. F. Hoffman
Keyword(s):  
Potassium Current ◽  
Binding Proteins ◽  
Membrane Conductance ◽  
Pipette Solution ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Gamma S

The acetylcholine-induced opening of potassium channels depends on GTP-binding proteins in the chick, guinea pig, frog, and rat. In contrast, Bubien and Woods (Biochem. Biophys. Res. Commun. 142: 1039-1045, 1987) have recently postulated that the acetylcholine response in cultured canine atrial cells may be independent of GTP-binding proteins. In whole cell patch-clamp experiments using cultured canine atrial cells, we did not detect an effect of GTP (10(-4) M) in the pipette solution on the acetylcholine-induced potassium current. However, 500 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) in the pipette diminished the response to acetylcholine. Pertussis toxin (30 ng/ml for 24 h) blocked the response to acetylcholine. With guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 3 microM) in the patch pipette, acetylcholine irreversibly increased membrane conductance. The current-voltage relationship for the persistently activated current was similar to that induced by acetylcholine. We conclude that the acetylcholine-induced potassium current in canine atrial cells behaves like that seen in other species and depends on GTP-binding proteins.

scholarly journals Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

1993 ◽  
Vol 120 (1) ◽  
pp. 37-45 ◽  
Author(s):  
L L Carter ◽  
T E Redelmeier ◽  
L A Woollenweber ◽  
S L Schmid
Keyword(s):  
Protein Function ◽  
Binding Proteins ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Vesicle Budding ◽  
Gtp Binding Proteins ◽  
Receptor Mediated Endocytosis ◽  
Gtp Binding ◽  
Gamma S

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

scholarly journals A Ypt32p Exchange Factor Is a Putative Effector of Ypt1p

2002 ◽  
Vol 13 (9) ◽  
pp. 3336-3343 ◽  
Author(s):  
Wei Wang ◽  
Susan Ferro-Novick
Keyword(s):  
Secretory Pathway ◽  
Binding Proteins ◽  
Functional Relationship ◽  
Membrane Traffic ◽  
Genetic Studies ◽  
Vesicle Tethering ◽  
Gtp Binding Proteins ◽  
Exchange Factor ◽  
Gtp Binding ◽  
Signal Cascade

Ypt1p regulates vesicle tethering and fusion events from the ER to the Golgi and through the early Golgi. Genetic studies have suggested a functional relationship between Ypt1p and Ypt31p/Ypt32p. Ypt31p and Ypt32p are a pair of functionally redundant GTPases that act after Ypt1p to mediate intra-Golgi traffic or the budding of post-Golgi vesicles from the trans-Golgi. Here we report that a novel Ypt32p exchange factor is a putative effector of Ypt1p. These findings implicate small GTP-binding proteins of the Ypt/Rab family in a signal cascade that directs membrane traffic through the secretory pathway.

scholarly journals Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes.

1992 ◽  
Vol 119 (5) ◽  
pp. 1077-1096 ◽  
Author(s):  
R Schwaninger ◽  
H Plutner ◽  
G M Bokoch ◽  
W E Balch
Keyword(s):  
G Protein ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Protein S ◽  
Rab Proteins ◽  
Heterotrimeric G Protein ◽  
Gtp Binding Proteins ◽  
Initial Event ◽  
Gtp Binding ◽  
Transport Vesicle

Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.

Fluoroaluminate- and GTP gamma S-induced stress, shortening, and myosin phosphorylation in airway smooth muscle

1993 ◽  
Vol 265 (1) ◽  
pp. L73-L79
Author(s):  
C. M. Hai ◽  
C. B. Ma
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Binding Proteins ◽  
Muscle Length ◽  
Active Stress ◽  
Myosin Phosphorylation ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Muscle Shortening ◽  
Gamma S

GTP-binding proteins in bovine tracheal smooth muscle were activated by fluoroaluminate and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and the sensitivities of fluoroaluminate- and GTP gamma S-induced active stress and myosin phosphorylation to muscle shortening were compared. Relative to the value of myosin phosphorylation at L0, unloaded shortening induced a 63% decrease in fluoroaluminate-activated steady-state myosin phosphorylation, but had no significant effect on GTP gamma S-activated myosin phosphorylation. These results were consistent with the hypothesis that shortening-sensitive and shortening-insensitive signal-transduction pathways coexist in airway smooth muscle. However, unlike myosin phosphorylation, active stress induced by fluoroaluminate was actually less sensitive to shortening. The amount of shortening necessary to reduce active stress to half of that at Lo was 65% in fluoroaluminate-activated tissues, but was only 34% in GTP gamma S-activated tissues. The observation of different sensitivities of fluoroaluminate-activated myosin phosphorylation and active stress suggests that GTP-binding proteins modulate the dependence of active stress on muscle length in smooth muscle.

Small GTP-binding protein, Rab6, is associated with secretory granules in atrial myocytes

1997 ◽  
Vol 272 (5) ◽  
pp. C1594-C1601 ◽  
Author(s):  
H. Iida ◽  
S. Tanaka ◽  
Y. Shibata
Keyword(s):  
Electron Microscopy ◽  
Intracellular Transport ◽  
Binding Proteins ◽  
Secretory Granules ◽  
Immunogold Electron Microscopy ◽  
Rab Proteins ◽  
Atrial Myocytes ◽  
Vesicular Traffic ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Rab proteins, a subfamily of small GTP-binding proteins, have been shown to play key roles in regulation of vesicular traffic in eukaryotic cells. In this study, we have intended to identify, the atrial granule-associated Rab proteins that seem to be required for formation or intracellular transport of the granules. Atrial granules contained at least four small GTP-binding proteins, and we have demonstrated by biochemical analysis that one of the small GTP-binding proteins associated with the atrial granules is a Rab6 protein (Rab6p). Rab6p was also detected in highly purified zymogen granules of pancreatic exocrine gland. Immunogold electron microscopy performed on ultrathin cryosections of rat auricle revealed that Rab6p was associated with the atrial granule membranes. Association of Rab6p with the atrial granule membranes was also confirmed by immunodiffusion electron microscopy in agarose-embedded atrial granules. These data indicate that Rab6p is associated with the atrial granules and that it might function in the intracellular traffic of the secretory granules in the atrial myocytes.

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