Isolated Plasma Membrane Vesicles Research Articles

Membrane phase separation drives organization and activation at bcr signaling complexes

Isolated plasma membranes separate into two coexisting liquid phases with distinct lipid and protein compositions but live cell plasma membranes do not macroscopically phase separate, leading to questions of whether and how the membrane phase transition contributes to functional heterogeneity in cells. Using quantitative super resolution microscopy we show that B cell receptor signaling platforms are nanoscale domains that quantitatively enrich membrane probes based on probe phase partitioning in isolated plasma membrane vesicles.

Exploring a role for the proximity to the plasma membrane miscibility phase transition in the gating of two temperature sensing channels

The Transient Receptor Potential (TRP) family channels TRPM8 and TRPV1 are well characterized temperature-gated ion channels whose functions are impacted by established ligands and a broad range of hydrophobic small molecules. For example, past work shows that ethanol lowers the gating temperature for both channels. Ethanol has also been shown to reduce the miscibility phase transition temperature in isolated plasma membrane vesicles isolated from mammalian membranes. This poster will present early investigations aimed at testing the hypothesis that the membrane phase transition influences the gating temperature of these channels, either directly by manipulating domain structure, or indirectly by impacting abundance and/or availability of allosteric regulators of channel function.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport.

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

Pinning Cholesterol Chemical Potential Impacts the Miscibility Transition in Isolated Plasma Membrane Vesicles

Giant plasma membrane vesicles (GPMVs) isolated from mammalian cell lines separate into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases below growth temperatures, and are often considered a model system for understanding lipid raft heterogeneity in intact cells. Past work shows GPMV transition temperature can be dependent on cell type, growth conditions, or the presence of small molecules, and the surface fraction of phases can be altered by modulating cholesterol levels in cells prior to GPMV isolation using methyl β cyclodextrin (MβCD). Past experiments have treated GPMVs as closed systems with fixed composition since lipids and membrane bound proteins are not soluble in the aqueous phase. It is possible to prepare GPMVs under conditions where cholesterol can exchange between vesicles and the aqueous phase, allowing us to examine vesicle thermodynamic behavior under conditions of fixed cholesterol chemical potential. Experimentally, this is accomplished by bathing vesicles in buffers containing MβCD and MβCD-cholesterol complexes. Recently published work has taken this approach to characterize and manipulate cholesterol chemical potential in intact cells (Ayuyan and Cohen BJ 114(4):904 2018) where it was shown that cholesterol chemical potential varies with cell density. Cell density also alters miscibility temperatures in GPMVs, leading us to speculate possible connections. This poster will present our ongoing work exploring the miscibility transition in GPMVs under conditions of fixed cholesterol chemical potential. We are exploring how the chemical potential of cholesterol within GPMVs varies with growth conditions, while examining how miscibility transition temperatures, surface fraction of phases, and the emergence of gel phase domains are impacted by varying cholesterol chemical potential. Our long term goal is to better understand how the miscibility transition couples to cholesterol chemical potential since this quantity governs binding between cholesterol and membrane bound proteins.

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

RBL-2H3 Proliferation is Modulated by Treatments that Shift Transition Temperatures in Isolated Plasma Membrane Vesicles

Giant plasma membrane vesicles (GPMVs) isolated from a variety of cell types appear homogeneous at physiological temperatures but separate into distinct liquid-ordered and liquid-disordered phases below their transition temperature (Tmix). Previous work in the lab has shown that Tmix is higher in GPMVs isolated from sparsely plated cells than those plated at a higher density (Gray et al. PLoS One 10(9):e0137741 (2015)). Since these cells are also contact inhibited, we hypothesize that differences in Tmix are related to their different growth rates. This previous study also found that Tmix is elevated in GPMVs isolated from cells preparing to undergo cell division compared to cells in other phases of the cell cycle. Based on these past results, we hypothesized that compounds which shift GPMV Tmix also affect the rate of cell growth when incubated with intact cells. To test this hypothesis, we incubated intact RBL-2H3 cells in n-alcohols and combinations of n-alcohols which we have previously shown to alter Tmix when added to isolated GPMVs (Machta et al. Biophys. J. 111(3):537-45 (2016)). We find that these treatments also alter cell growth as measured by counting cells present after 24h of incubation with compounds in complete media at 37°C. Specifically, fewer cells are counted in dishes containing n-alcohols that lower Tmix in GPMVs than that of a control, whereas an even greater number of cells are counted in dishes containing an n-alcohol that raises Tmix in GPMVs. In addition, we find that cells adapt their membrane composition to compensate for the effect of n-alcohols on GPMV Tmix when incubated with n-alcohols for 24h. Ongoing experiments are being conducted to illuminate the biochemical pathways underlying the observed changes in cell proliferation.

Membrane Transition Temperature Determines Cisplatin Response.

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients.

Plasma membrane vesicles decorated with glycolipid-anchored antigens and adjuvants via protein transfer as an antigen delivery platform for inhibition of tumor growth

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity.

Hexadecanol Reverses Ethanol Induced Tadpole Anesthesia and Raises Critical Temperatures in Isolated Plasma Membrane Vesicles

Many small molecules induce general anesthesia in animals ranging from insects to mammals. Although their mechanism of action remains controversial, it is known that a compound's efficacy as an anesthetic is strongly predicted by both its hydrophobicity and its potency in inhibiting certain ligand gated ion channels. We recently published experiments showing that n-alcohol anesthetics (n≤10) take plasma membrane derived vesicles away from a biologically tuned, two dimensional (2D) liquid-liquid critical point [1]. Here, we present one line of investigation into our working hypothesis that anesthetics exert at least some of their effects by interfering with native regulation by the nearly critical plasma membrane. While most compounds, including the n≤10-alcohol anesthetics, lower critical temperatures in membranes, certain compounds raise critical temperatures counteracting the effects of anesthetics on membrane criticality. These compounds should specifically reverse effects mediated through membrane criticality. We demonstrate that hexadecanol (n=16) is such a compound, raising critical temperatures in cell-derived vesicles in a dose-dependent manner. We also present evidence that hexadecanol reverses ethanol's anesthetic effects on the tadpole righting response, in a manner quantitatively predicted by its effects on membrane criticality.1. Gray, E.; Karslake, J.; Machta, B.B.; Veatch, S.L.; Biophys. J. 105(12): 2751-9 (2013).

Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress.

Size and Acylation Influence the Lateral Mobility of Plasma Membrane Proteins in Live Cells

Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mammalian cells. Using single fluorescent particle tracking, we quantify diffusion parameters of a large panel of fluorescent fusion membrane proteins ranging in size, mode of membrane anchoring, and putative phase-association. These include palmitoylated or non-palmitoylated versions of three transmembrane proteins (truncated linker of activated T-cell, truncated haemagglutinin, and β2 adrenergic receptor) as well as three proteins anchored with lipid moieties (GPI, palmitoyl and myristoyl, or geranylgeranyl). We present an analysis that utilizes Brownian simulations to aid in interpreting heterogeneity. With the exception of two of the palmitoylated transmembrane proteins, diffusion of all constructs is unconfined and consistent with Brownian motion at 37°C at the time-scales investigated (20 ms-1 sec). We explore the contributions of lipid mixing to confinement by modulating cholesterol levels and through the use of biochemical perturbations that affect the temperature of the immiscibility phase transition in isolated plasma membrane vesicles. Among our findings is that diffusion and confinement are highly temperature sensitive. Furthermore, our results indicate a complicated size-dependence of diffusion suggesting that diffusion of small probes is particularly sensitive to dimerization when it occurs in either a biological context or due to labeling techniques. Overall, we explore the contribution of phase-mediated membrane heterogeneity to protein mobility while highlighting several factors that can complicate the interpretation of lateral diffusion data.

Polyamines Depolarize the Membrane and Initiate a Cross-Talk Between Plasma Membrane Ca2+ and H+ Pumps

There are multiple studies, exploring effects of polyamines on cation and K+ channels in animal and plant cells, but much less is known on their potential effects on cation-transporting ATPases. In this work non-invasive MIFE and conventional microelectrode techniques were applied to study the effects of polyamines (PAs) on Ca2+ and H+ transport and membrane potential in living pea roots. Putrescine (Put2+) or spermine (Spm4+) in 1 mM concentration caused a large increase of Ca2+ efflux across plasma membrane (PM), sensitive to Ca2+ pump inhibitor eosine yellow (EY) but insensitive to amiloride. Put2+ and Spm4+ caused contrasting effects on H+ fluxes: induced net H+ efflux (due to activity of the PM H+ pump) and influx, respectively. Use of EY and vanadate, non-specific inhibitor of P-type pumps, converted Spm4+-induced H+ influx to transient H+ efflux and suppressed H+ fluxes, respectively. The former result was consistent with Ca2+/H+ antiport activity of the PM Ca2+ pump. However, EY also reduced Put2+-induced H+ efflux. The latter result may be explained by down-regulation of the H+ pump activity by increased intracellular Ca2+. When it comes to direct effect of PAs on the H+ pump in isolated PM vesicles, Put2+ did not produce any effect and Spm4+ partly suppressed H+ pumping. All PAs, Spm4+>Spd3+>Put2+ caused substantial (up to 70 mV) membrane depolarization, abolished by Gd3+ and strongly decelerated by removal of external Ca2+. Analysis of net ion fluxes, induced by different PAs, led us to a conclusion that transport of PAs themselves directly contributes to membrane potential changes. Therefore, PAs induce Ca2+ pump activity and differentially affect H+ fluxes and electric potential difference across the PM. Supported by CONACyT and ARC grants and by University of Tasmania visiting professorship to IP.

Long term myriocin treatment increases MRP1 transport activity

We investigated the effect of myriocin treatment, which extensively depletes sphingolipids from cells, on multidrug resistance-related protein 1 (MRP1) efflux activity in MRP1 expressing cells and isolated plasma membrane vesicles. Our data reveal that both short term (3 days) and long term (7 days) treatment effectively reduce the cellular sphingolipid content to the same level. Intriguingly, a two-fold increase in MRP1-mediated efflux activity was observed following long term treatment, while short term treatment had no impact. Very similar data were obtained with plasma membrane vesicles isolated from myriocin-treated cells. Exploiting the cell-free vesicle system, Michaelis–Menten analysis revealed that the intrinsic MRP1 activity remained unaltered; however, the fraction of active transporter molecules increased. We demonstrate that the latter effect is due to an enhanced recruitment of MRP1 into lipid raft fractions, thereby promoting MRP1 activity.

Transport and bioactivity of cyanidin 3-glucoside into the vascular endothelium

Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC No. 2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926) and on an ischemia–reperfusion model in isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, despite their limited oral bioavailability and very low postabsorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor in their protective activity on endothelial function.

Using Cell Surface Protein Distributions To Investigate The Physical Basis Of Plasma Membrane Lateral Heterogeneity

It is widely recognized that proteins and lipids are heterogeneously distributed on the cell surface, yet little is known regarding the underlying physical principles that give rise to this membrane organization. Recently, we characterized robust and dynamic critical fluctuations in isolated plasma membrane vesicles and proposed that critical fluctuations provide a plausible physical basis for 50nm), consistent with previous studies. In addition, the detailed shapes of experimentally derived correlation functions provide additional information into the organizing principles that give rise to membrane heterogeneity. We evaluate the validity of different proposed mechanisms of membrane organization by fitting our experimental findings to predictions of various models. Models investigated include critical fluctuations, micro-emulsions, and membrane coupling to cytoskeletal components. Implications for cellular processes such as signaling will be discussed.1. Veatch, S. L., P. Cicuta, P. Sengupta, A. Honerkamp-Smith, D. Holowka, and B. Baird. 2008. Critical fluctuations in plasma membrane vesicles. ACS Chemical Biology 3:287-293.

Use Of Isolated Vesicles From Xenopus Oocytes For Planar Patch Clamp Recordings

Oocytes from the Xenopus laevis toad represent an efficient expression system. The two-electrode voltage-clamp technique (TEVC) as well as patch clamp recordings from excised patches are powerful tools to study ion channels following expression in Xenopus oocytes. The TEVC technique allows the measurement of macroscopic currents from whole cells. It is widely used in basic research and also applied for compound screening in pharmaceutical industry. Recordings from excised patches enable the analysis of ion channels under cell free conditions and even single channel analysis. In contrast to the TEVC technique, its application requires highly skilled electrophysiologists.A publication by Yong Zhang et al. ∗ describes a technique to prepare isolated membrane vesicles from Xenopus oocytes by incubating them, after removal of the vitelline membrane, in a hypertonic “blebbing” solution. This treatment induces the formation of plasma membrane blebs that eventually detach from the oocyte surface to form isolated plasma membrane vesicles (PMVs).Here we used these PMVs to create macro patches onto a planar borosilicate glass surface using the Port-a-Patch (Nanion) automated patch clamp device. The PMVs formed readily gigaseals and showed small capacities as expected for macro patches. Currents could be recorded from voltage-gated as well as ligand-gated ion channels.In conclusion, we present an application that has the potential to overcome the practical limitations associated with the generation of excised patches and might enable the automated analysis of such patches.∗Mechanically gated channel activity in cytoskeleton-deficient plasma membrane blebs and vesicles from Xenopus oocytes.Yong Zhang, Feng Gao, Vsevolod L. Popov,∗ Julie W. Wen,∗ and Owen P. Hamill J Physiol. 2000 February 15; 523(Pt 1): 117-130.

A fruit‐specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria × ananassa) fruit ripening

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria xananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.

ATP‐ and growth substance‐dependent cell‐free enlargement of plasma membrane vesicles from soybean

Growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans prepared by aqueous two-phase partition and everted by freezing and thawing has been achieved in a cell-free system. In the presence of 100 microM ATP in 40 mM HEPES buffer, pH 7, enlargement of isolated plasma membrane vesicles was accelerated by the synthetic plant growth factor, 2,4-dichlorophenoxyacetic acid (2,4-D), compared to ATP alone, 2,4-D alone or no additions. After 20 min with 1 microM 2,4-D, vesicles increased in diameter, 20% on average. Although vesicle diameters in the presence or absence of 2,4-D overlapped, the means were clearly separated. The 20% increases in diameter corresponded to a doubling of vesicle volume. Both 100 microM ATP and 1 microM 2,4-D were necessary to stimulate the cell-free vesicle enlargement. In the presence of 1 microM 2,4-D, enlargement observed with 100 microM ATP was greater than with either 10 microM ATP or 500 microM ATP alone. In the presence of 100 microM ATP, vesicle enlargement was proportional to the logarithm of 2,4-D concentration. With the growth-inactive 2,4-D analog, 2,3-D, no vesicle enlargement was observed either alone or in the presence of 100 microM ATP. Right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination suggesting that the responsible ATP site was on the inside of the cell.

Expression of yeast SOD2 in transgenic rice results in increased salt tolerance

Agricultural productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to remove sodium ions from the cytosol via plasma membrane Na +/H + antiporters. In the present study, we demonstrated that expressing the plasma membrane Na +/H + antiporter SOD2 from yeast ( Schizosaccharomyces pombe) in transgenic rice increased salt tolerance. These transgenic plants accumulated more K +, Ca 2+, Mg 2+ and less Na + in their shoots compared with those of non-transformed controls. Moreover, measurements on isolated plasma membrane vesicles derived from the SOD2 transgenic rice plant roots showed that the vesicles had enhanced P-ATPase hydrolytic activity. Furthermore, the transformed rice plants maintained higher levels of photosynthesis and root proton exportation capacity, whereas reduced ROS generation. Physiological analysis suggested that transgenic rice plants might employ multiple mechanisms to improve their salt tolerance under salt stress conditions.