Chemically induced vesiculation as a platform for studying TMEM16F activity

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

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TMEM16F is a Calcium-Activated Phospholipid Scramblase Required for Chemically-Induced Giant Plasma Membrane Vesicles

Biophysical Journal ◽

10.1016/j.bpj.2017.11.3335 ◽

2018 ◽

Vol 114 (3) ◽

pp. 610a

Author(s):

Tina W. Han ◽

Wenlei Ye ◽

Neville P. Bethel ◽

Mario Zubia ◽

Michael Grabe ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Phospholipid Scramblase ◽

Chemically Induced

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ATP-dependent transport of glutathione conjugate of 7β,8α-dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles

Biochemical Journal ◽

10.1042/bj3320799 ◽

1998 ◽

Vol 332 (3) ◽

pp. 799-805 ◽

Cited By ~ 11

Author(s):

Sanjay K. SRIVASTAVA ◽

Xun HU ◽

Hong XIA ◽

Richard J. BLEICHER ◽

Howard A. ZAREN ◽

...

Keyword(s):

Plasma Membrane ◽

Atp Hydrolysis ◽

Membrane Vesicles ◽

Competitive Inhibitor ◽

Dependent Manner ◽

Plasma Membrane Vesicles ◽

High Affinity ◽

Dependent Transport ◽

Detoxification Pathway ◽

Stimulation Of

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 µM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis–Menten kinetics (Km 46 µM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis–Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 µM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 µM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 µM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

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Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

10.1101/731364 ◽

2019 ◽

Cited By ~ 2

Author(s):

AD Skinkle ◽

I Levental

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Permeability Barrier ◽

Hydrophobic Core ◽

Plasma Membrane Vesicles ◽

Giant Vesicles ◽

Membrane Asymmetry ◽

Preparation Conditions ◽

Biophysical Analysis ◽

Membrane Models

ABSTRACTGiant Plasma Membrane Vesicles (GPMVs) are a widely used model system for biochemical and biophysical analysis of the isolated mammalian plasma membrane (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed; i.e. that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles (GUVs). We attribute this leakiness to relatively large and heterogeneous pores formed by rupture of vesicles from cells. These pores are stable and persist throughout experimentally relevant time scales. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are of critical importance for interpreting experiments utilizing GPMVs as plasma membrane models, particularly for drug permeation and membrane asymmetry.STATEMENT OF SIGNIFICANCEA critical assumption in using Giant Plasma Membrane Vesicles to study membrane penetration and interactions is that these vesicles maintain the permeability barrier of the native membrane from which they form. Using large fluorescently-labeled hydrophilic probes, we demonstrate that this assumption is often incorrect and conclude that macromolecular solutes permeate GPMVs through stable pores formed during shear-induced rupture of vesicles from cells. Using these insights into the mechanisms of poration, we demonstrate an approach to isolate sealed GPMVs.

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Inhibition of Maize (Zea mays L.) Root Plasma Membrane-Bound Redox Activities by Coumarins

Zeitschrift für Naturforschung C ◽

10.1515/znc-1994-7-809 ◽

1994 ◽

Vol 49 (7-8) ◽

pp. 447-452 ◽

Cited By ~ 10

Author(s):

Sabine Lüthje ◽

José A. Gonzaléz-Reyes ◽

Placido Navas ◽

Olaf Döring ◽

Michael Böttger

Keyword(s):

Zea Mays ◽

Plasma Membrane ◽

Zea Mays L ◽

Membrane Vesicles ◽

Dependent Manner ◽

Plasma Membrane Vesicles ◽

Two Phase ◽

Concentration Dependent Manner ◽

Oxidoreductase Activity ◽

Membrane Bound

Modulation of plasma membrane-bound NADH:hexacyanoferrate III oxidoreductase activities by dicumarol and warfarin was investigated with plasma membrane vesicles of Zea mays L. (cv. Sil Anjou 18) roots, prepared by aqueous two phase partitioning. Vesicles were about 65% right-side out orientated as demonstrated by enzyme latency of vanadate sensitive ATPase activity. Dicumarol or warfarin, respectively, inhibited NADH:hexacyanoferrate III oxidoreductase activity in a concentration-dependent manner and inhibition could be reversed partially by addition of quinones

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High-affinity pH-dependent passive Ca binding by myometrial plasma membrane vesicles

AJP Cell Physiology ◽

10.1152/ajpcell.1983.244.1.c61 ◽

1983 ◽

Vol 244 (1) ◽

pp. C61-C67 ◽

Cited By ~ 18

Author(s):

A. K. Grover ◽

C. Y. Kwan ◽

E. E. Daniel

Keyword(s):

Plasma Membrane ◽

Reaction Medium ◽

Membrane Vesicles ◽

Hill Coefficient ◽

Dependent Manner ◽

Plasma Membrane Vesicles ◽

High Affinity ◽

Ph Dependent ◽

The Difference ◽

Ph Buffer

Rat myometrium plasma membrane-(PM) enriched fraction N1 binds calcium passively in a pH-dependent manner at a Ca2+ concentration of 1 microM. The Ca binding increases with increasing pH from 6.27 to 7.47 with a half maximum near 6.8. The difference between binding at 6.27 and 7.07 (the pH-dependent Ca binding) depends on the pH of the reaction medium rather than the pH of the medium in which the membranes had previously been suspended. The pH-dependent Ca binding is not an artifact due to EGTA, the pH buffer used, or soluble protein trapped inside the membrane vesicles. The pH-dependent Ca binding occurs with a dissociation constant value of 0.28 microM and Hill coefficient of 2.37 for Ca2+. The high affinity pH-dependent Ca uptake and the release of Ca2+ from the membranes is virtually complete in 10 s in the presence of 1 microM A23187 but not in its absence. The distribution of the pH-dependent Ca binding in the various rat myometrium subcellular fractions parallels the activity of 5'-nucleotidase in these fractions and not the activities of NADPH-dependent or succinate-dependent cytochrome c reductases. The high affinity and rapid binding and release of Ca at the pH-dependent Ca binding sites in the PM-enriched fraction suggests that the binding and release from these sites may play a key role in excitation-contraction coupling of the smooth muscle.

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Chemically induced plasma membrane vesicles as a useful tool for the investigation of virus binding to susceptible cells

Journal of Virological Methods ◽

10.1016/0166-0934(93)90028-p ◽

1993 ◽

Vol 42 (2-3) ◽

pp. 147-154 ◽

Cited By ~ 2

Author(s):

René Julen ◽

Ulrich N. Wiesmann ◽

Hans Koblet

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Virus Binding ◽

Chemically Induced

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Ceramide and sphingosine have an antagonistic effect on the plasma-membrane Ca2+-ATPase from human erythrocytes

Biochemical Journal ◽

10.1042/bj3620247 ◽

2002 ◽

Vol 362 (2) ◽

pp. 247-251 ◽

Cited By ~ 18

Author(s):

Claudia COLINA ◽

Vincenza CERVINO ◽

Gustavo BENAIM

Keyword(s):

Plasma Membrane ◽

Second Messengers ◽

Membrane Vesicles ◽

Inhibitory Effect ◽

Antagonistic Effect ◽

Dependent Manner ◽

Plasma Membrane Vesicles ◽

Key Enzyme ◽

Hydroxy Groups ◽

Dose Dependent

The plasma-membrane Ca2+-ATPase is a key enzyme in the regulation of the intracellular Ca2+ concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca2+. In view of the fact that the Ca2+-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca2+-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca2+ and the Vmax of the enzyme. Furthermore, this second messenger also stimulates Ca2+ transport in inside—out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca2+-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca2+-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca2+ concentration.

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