scholarly journals Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices

1992 ◽  
Vol 117 (6) ◽  
pp. 1181-1196 ◽  
Author(s):  
LJ Robinson ◽  
S Pang ◽  
DS Harris ◽  
J Heuser ◽  
DE James
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Protein S ◽  
Confocal Laser ◽  
Intact Cells ◽  
Intracellular Location ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Gamma S

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.

scholarly journals Activated release of membrane-anchored TGF-alpha in the absence of cytosol

1993 ◽  
Vol 122 (1) ◽  
pp. 95-101 ◽  
Author(s):  
MW Bosenberg ◽  
A Pandiella ◽  
J Massagué
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Atp Hydrolysis ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Vesicular Traffic ◽  
Kinase C ◽  
Streptolysin O ◽  
Independent Protein

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA-sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.

scholarly journals Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells.

1987 ◽  
Vol 105 (1) ◽  
pp. 191-197 ◽  
Author(s):  
T W Howell ◽  
S Cockcroft ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Rank Order ◽  
Regulatory Protein ◽  
Metabolic Inhibitors ◽  
Pyrimidine Nucleotides ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Gamma S ◽  
Sufficient Stimulus ◽  
Rat Mast Cells

Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP greater than XTP greater than GTP much greater than ATP. Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca2+ plus ITP inhibit secretion in the order 2'-deoxyGDP greater than GDP greater than o-GDP greater than ADP approximately equal to 2'deoxyADP approximately equal to IDP. Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-gamma-S greater than GppNHp greater than GppCH2p) which synergize with Ca2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.

scholarly journals A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation.

1991 ◽  
Vol 115 (5) ◽  
pp. 1225-1236 ◽  
Author(s):  
F J Stafford ◽  
J S Bonifacino
Keyword(s):  
Protein Degradation ◽  
Secretory Pathway ◽  
Fibroblast Cell ◽  
Cell System ◽  
Current Evidence ◽  
Permeabilized Cell ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
A Site

Analysis of the fate of a variety of newly synthesized proteins in the secretory pathway has provided evidence for the existence of a novel protein degradation system distinct from that of the lysosome. Although current evidence suggests that proteins degraded by this system are localized to a pre-Golgi compartment before degradation, the site of proteolysis has not been determined. A permeabilized cell system was developed to examine whether degradation by this pathway required transport out of the ER, and to define the biochemical characteristics of this process. Studies were performed on fibroblast cell lines expressing proteins known to be sensitive substrates for this degradative process, such as the chimeric integral membrane proteins, Tac-TCR alpha and Tac-TCR beta. By immunofluorescence microscopy, these proteins were found to be localized to the ER. Treatment with cycloheximide resulted in the progressive disappearance of intracellular staining without change in the ER localization of the chimeric proteins. Cells permeabilized with the pore-forming toxin streptolysin O were able to degrade these newly synthesized proteins. The protein degradation seen in permeabilized cells was representative of that seen in intact cells, as judged by the similar speed of degradation, substrate selectivity, temperature dependence, and involvement of free sulfhydryl groups. Degradation of these proteins in permeabilized cells took place in the absence of transport between the ER and the Golgi system. Moreover, degradation occurred in the absence of added ATP or cytosol, and in the presence of apyrase, GTP gamma S, or EDTA; i.e., under conditions which prevent transport of proteins out of the ER. The efficiency and selectivity of degradation of newly synthesized proteins were also conserved in an isolated ER fraction. These data indicate that the machinery responsible for pre-Golgi degradation of newly synthesized proteins exists within the ER itself, and can operate independent of exogenously added ATP and cytosolic factors.

scholarly journals Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4

1995 ◽  
Vol 309 (3) ◽  
pp. 813-823 ◽  
Author(s):  
K Ishii ◽  
H Hayashi ◽  
M Todaka ◽  
S Kamohara ◽  
F Kanai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Terminal Region ◽  
Target Cells ◽  
Glut4 Translocation ◽  
Intracellular Loop ◽  
Intact Cells ◽  
Intracellular Targeting

Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We constructed c-myc epitope-tagged glucose transporter type 1 (GLUT1myc) and found that the GLUT1myc was also translocated to the cell surface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was less than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin-stimulated translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intracellular loop and cytoplasmic C-terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation.

scholarly journals Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

1998 ◽  
Vol 333 (3) ◽  
pp. 779-786 ◽  
Author(s):  
Jan Willem KOK ◽  
Teresa BABIA ◽  
Karin KLAPPE ◽  
Gustavo EGEA ◽  
Dick HOEKSTRA
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Incubation Temperature ◽  
De Novo ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Vesicular Stomatitis ◽  
Glycoprotein Transport

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 °C, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.

scholarly journals Differential effects of G-protein activators on 5-hydroxytryptamine and platelet-derived growth factor release from streptolysin-O-permeabilized human platelets

1996 ◽  
Vol 314 (1) ◽  
pp. 123-128 ◽  
Author(s):  
Philip J. PADFIELD ◽  
Ninder PANESAR ◽  
Patricia HENDERSON ◽  
Joseph J. BALDASSARE
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Proteins ◽  
Protein S ◽  
Human Platelets ◽  
Dense Core ◽  
Platelet Derived Growth Factor ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Dense Core Granules

In this paper we have used streptolysin O (SLO)-permeabilized human platelets to examine the G-protein(s) that control Ca2+-independent secretion from α and dense-core granules. As shown for electropermeabilized platelets, Ca2+ alone stimulated a concentration-dependent increase in 5-hydroxytryptamine (5-HT) (dense-core-granule marker) and platelet-derived growth factor (PDGF) (α-granule marker) release from the SLO-permeabilized cells. The EC50 values for Ca2+-dependent 5-HT and PDGF release were 5 μM and 10 μM respectively. Guanosine 5´-[γ-thio]triphosphate (GTP[S]) (100 μM) stimulated Ca2+-independent release from both α and dense-core granules. In contrast, AlF4- had no effect on Ca2+-independent release from either α or dense-core granules. Neither GTP[S] nor AlF4- appeared to have a significant effect on Ca2+-dependent release from α and dense-core granules. GTP[S] can activate both heterotrimeric and low-molecular-mass G-proteins, whereas AlF4- activates only heterotrimeric G-proteins. Our results, therefore suggest that secretion in the human platelet is regulated by a small G-protein. Both GTP[S]- and Ca2+-dependent secretion were effected by extending the time between permeabilization with SLO and stimulation of secretion. GTP[S]-stimulated secretion from α and dense-core granules decreased rapidly after permeabilization. In contrast, Ca2+-dependent 5-HT and PDGF release ran down at a much lower rate. These observations indicate that GTP[S] and Ca2+ act through parallel pathways to stimulate secretion from SLO-permeabilized platelets.

scholarly journals Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O

1988 ◽  
Vol 250 (2) ◽  
pp. 375-382 ◽  
Author(s):  
J Stutchfield ◽  
S Cockcroft
Keyword(s):  
Rank Order ◽  
Guanine Nucleotides ◽  
Hl60 Cell ◽  
Maximal Response ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Competitive Inhibitors ◽  
In Series ◽  
Gamma S

The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5′-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5′-[beta gamma-imido]-triphosphate greater than guanosine 5′-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.

scholarly journals Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

1991 ◽  
Vol 112 (1) ◽  
pp. 39-54 ◽  
Author(s):  
S G Miller ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Secretory Granules ◽  
Intact Cells ◽  
Transport Reaction ◽  
Permeabilized Cells ◽  
Transmembrane Glycoprotein ◽  
Transport Vesicles ◽  
Constitutive Secretion

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).

Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes

2000 ◽  
Vol 113 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Z.A. Khayat ◽  
P. Tong ◽  
K. Yaworsky ◽  
R.J. Bloch ◽  
A. Klip
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Surface Membrane ◽  
Dorsal Surface ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
L6 Myotubes ◽  
Triton X ◽  
Phosphatidylinositol 3

We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.

scholarly journals Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

1996 ◽  
Vol 7 (3) ◽  
pp. 397-408 ◽  
Author(s):  
A J O'Sullivan ◽  
A M Brown ◽  
H N Freeman ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Peptide Sequencing ◽  
Bovine Brain ◽  
Nucleotide Exchange ◽  
Permeabilized Cells ◽  
Guanine Nucleotide Exchange ◽  
Exchange Inhibitor ◽  
Streptolysin O ◽  
Gamma S ◽  
Brain Cytosol

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

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