scholarly journals ATP-dependent transport of reduced glutathione in yeast secretory vesicles

1998 ◽  
Vol 334 (3) ◽  
pp. 723-729 ◽  
Author(s):  
James F. REBBEOR ◽  
Gregory C. CONNOLLY ◽  
Mark E. DUMONT ◽  
Nazzareno BALLATORI
Keyword(s):  
Direct Evidence ◽  
Reduced Glutathione ◽  
Membrane Vesicles ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Transport Mechanisms ◽  
Vesicle Membrane ◽  
Dependent Transport ◽  
Dependent Pathway ◽  
Taurocholate Transport

Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19±5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2,4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18±2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4,4´-di-isothiocyanatostilbene-2,2´-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.

scholarly journals Kiss-and-Coat and Compartment Mixing: Coupling Exocytosis to Signal Generation and Local Actin Assembly

2006 ◽  
Vol 17 (4) ◽  
pp. 1495-1502 ◽  
Author(s):  
Anna M. Sokac ◽  
William M. Bement
Keyword(s):  
Cell Types ◽  
Secretory Vesicle ◽  
Signal Generation ◽  
General Mechanism ◽  
Fusion Pore ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Vesicle Membrane ◽  
Regulated Exocytosis ◽  
Broad Variety

Regulated exocytosis is thought to occur either by “full fusion,” where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by “kiss-and-run,” where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed “kiss-and-coat,” which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.

scholarly journals Relative Contribution of PIN-containing Secretory Vesicles and Plasma Membrane PINs to the Directed Auxin Transport: Theoretical Estimation

2018 ◽  
Author(s):  
Sander Hille ◽  
Maria Akhmanova ◽  
Matouš Glanc ◽  
Alexander Johnson ◽  
Jiří Friml
Keyword(s):  
Plasma Membrane ◽  
Auxin Transport ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Transport Mechanisms ◽  
In Planta ◽  
Major Mechanism ◽  
Relative Contribution ◽  
Secretory Transport ◽  
Auxin Flux

Intercellular transport of auxin is driven by PIN-formed (PIN) proteins. PINs are localized at the plasma membrane (PM) and on constitutively recycling endomembrane vesicles. Therefore, PINs can mediate auxin transport either by direct translocation across the PM or by pumping it into secretory vesicles (SVs), leading to its secretory release upon fusion with the PM. Which of these two mechanisms dominates is a matter of debate. Here we addressed the issue with a mathematical modeling approach. We demonstrate that the efficiency of secretory transport depends on SV size, half-life of PINs on the PM, pH, exocytosis frequency and PIN density. 3D-SIM microscopy was used to determine PIN density on the PM. Combing this data with published values of the other parameters, we show that the transport activity of PINs in SVs would have to be at least 1000x greater than on the PM in order to produce a comparable macroscopic auxin transport. If both transport mechanisms operated simultaneously and PINs were equally active on SVs and PM, the contribution of secretion to the total auxin flux would be negligible. In conclusion, while secretory vesicle-mediated transport of auxin is intriguing and theoretically possible model, it unlikely to be a major mechanism of auxin transport in planta.

Molecular Machinery and Mechanism of Cell Secretion

2005 ◽  
Vol 230 (5) ◽  
pp. 307-319 ◽  
Author(s):  
Bhanu P. Jena
Keyword(s):  
Lipid Membrane ◽  
Secretory Vesicle ◽  
Secretory Process ◽  
Secretory Vesicles ◽  
Cell Secretion ◽  
Vesicle Membrane ◽  
Cellular Processes ◽  
Molecular Machinery ◽  
Secretory Products ◽  
First Time

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are Packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called poro-somes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamen-tal cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the Porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The Molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.

scholarly journals Taurocholate–sodium co-transport by brush-border membrane vesicles isolated from rat ileum

1978 ◽  
Vol 174 (3) ◽  
pp. 951-958 ◽  
Author(s):  
Heinrich Lücke ◽  
Gertraud Stange ◽  
Rolf Kinne ◽  
Heini Murer
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Precipitation Method ◽  
Electrical Potential Difference ◽  
Vesicle Membrane ◽  
Brush Border Membranes ◽  
Taurocholate Transport

Uptake of taurocholate into brush-border membrane vesicles isolated from rat small intestine by a Ca2+ -precipitation method was investigated by using a rapid-filtration technique. Uptake of taurocholate by ileal brush-border membranes consisted of three phenomena: binding to the outside of the vesicles, transfer across the vesicle membrane and binding to the intravesicular compartment. The transport of taurocholate across the brush-border membranes was stimulated in the presence of Na+ compared with the presence of K+; stimulation was about 11-fold in the presence of a NaCl gradient (Nao>Nai), where the subscripts refer to ‘outside’ and ‘inside’ respectively, and 4-fold under equilibrium conditions for Na+ (Nao=Nai). In the presence of a Na+ gradient a typical ‘overshoot’ phenomenon was observed. Membranes preloaded with unlabelled taurocholate showed an accelerated entry of labelled taurocholate (tracer exchange) in the presence of Na+ compared with the presence of K+. The stimulation by Na+ was observed only in membrane preparations from the ileum. Addition of monactin, an ionophore for univalent cations, decreased the Na+-gradient-driven taurocholate uptake. The Na+-dependent taurocholate transport showed saturation kinetics and the phenomenon of counterflow and was inhibited by glycocholate. Other cations such as Li+, Rb+ and Cs+ could not replace Na+ in its stimulatory action. When the electrical potential difference across the vesicle membrane was altered by establishing different diffusion potentials (anion replacement; K+ gradient±valinomycin) a more-negative potential inside stimulated Na+-dependent taurocholate transport. These data demonstrate the presence of a rheogenic (potential sensitive) Na+–taurocholate co-transport system in ileal brush-border membranes and support the hypothesis that the reabsorption of bile acids in the ileum is a secondary active uptake.

scholarly journals Secretory Vesicles Targeted to Plasma Membrane During Pollen Germination and Tube Growth

2021 ◽  
Vol 8 ◽  
Author(s):  
Huaqiang Ruan ◽  
Jiang Li ◽  
Ting Wang ◽  
Haiyun Ren
Keyword(s):  
Plasma Membrane ◽  
Pollen Tube ◽  
Pollen Germination ◽  
Higher Plants ◽  
Current Knowledge ◽  
Secretory Vesicle ◽  
Signaling Molecules ◽  
Tube Growth ◽  
Secretory Vesicles ◽  
Vesicle Membrane

Pollen germination and pollen tube growth are important biological events in the sexual reproduction of higher plants, during which a large number of vesicle trafficking and membrane fusion events occur. When secretory vesicles are transported via the F-actin network in proximity to the apex of the pollen tube, the secretory vesicles are tethered and fused to the plasma membrane by tethering factors and SNARE proteins, respectively. The coupling and uncoupling between the vesicle membrane and plasma membrane are also regulated by dynamic cytoskeleton, proteins, and signaling molecules, including small G proteins, calcium, and PIP2. In this review, we focus on the current knowledge regarding secretory vesicle delivery, tethering, and fusion during pollen germination and tube growth and summarize the progress in research on how regulators and signaling molecules participate in the above processes.

ATP-dependent GSH and glutathioneS-conjugate transport in skate liver: role of an Mrp functional homologue

2000 ◽  
Vol 279 (2) ◽  
pp. G417-G425 ◽  
Author(s):  
James F. Rebbeor ◽  
Gregory C. Connolly ◽  
John H. Henson ◽  
James L. Boyer ◽  
Nazzareno Ballatori
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Direct Evidence ◽  
Membrane Vesicles ◽  
Protein Band ◽  
Transport Activity ◽  
Homologous Protein ◽  
Liver Membrane ◽  
Associated Proteins ◽  
Dependent Transport

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate ( Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using 3H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant ( K m) = 32 ± 9 μM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of 3H-labeled S-ethylglutathione and 3H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway ( K m = 12 ± 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.

scholarly journals Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yali Liu ◽  
Ling Zhang ◽  
Shaofeng Wei ◽  
Jinyang Cai ◽  
Zhenzhong Zang ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Human Intestinal Epithelial Cells ◽  
Pulsatilla Chinensis ◽  
Dependent Transport ◽  
Cytotoxic Studies

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

The Orientation of Cytochrome c Oxidase in Coupled Phospholipid Membrane Vesicles—A Spin-Label Study

1975 ◽  
Vol 53 (3) ◽  
pp. 364-370 ◽  
Author(s):  
J. A. Kornblatt ◽  
W. L. Chen ◽  
J. C. Hsia ◽  
G. R. Williams
Keyword(s):  
Molecular Weight ◽  
Binding Site ◽  
Cytochrome Oxidase ◽  
Spin Label ◽  
Resonance Spectrum ◽  
Membrane Vesicles ◽  
Phospholipid Membrane ◽  
Vesicle Membrane ◽  
Exterior Surface ◽  
Label Study

Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. We have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-labeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim &Racker ((1972) J. Biol. Chem. 247, 1338–1339) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles. NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with [Formula: see text] of 1 min at 22 °C. The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.

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