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-ß-cyclodextrin (MßCD) 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 MßCD 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.

Involvement of plasma membrane H+-ATPase in diamide-induced extracellular alkalization by roots from pea seedlings.

MAIN CONCLUSION: The plasma membrane H+-ATPase can be considered as a redox-dependent enzyme, because diamide-mediated inhibition of its hydrolytic and transport activities is accompanied by alkalization of the rhizosphere and retardation of root growth. Plasma membranes were isolated from roots of etiolated pea seedlings treated in the presence of an oxidant-diamide and an inhibitor of redox-sensitive protein phosphatase-phenylarsine oxide. Hydrolytic and proton transport activities of H+-ATPase were determined. The effects of diamide appeared in inhibition of both ATP hydrolysis and the proton transport. However, root treatment with phenylarsine oxide only slightly reduced Vmax, but did not affect ATP-dependent proton transport. The thiol groups of cysteines in the proteins can act as molecular targets for both compounds. However, treatment of isolated membranes with diamide or dithiothreitol did not have any effect on the H+ transport. It can be assumed that water-soluble diamide acts indirectly and its effects are not associated with oxidation of H+-ATPase cysteines. Therefore, plasmalemma was subjected to PEGylation-process where reduced cysteines available for PEG maleimide (5 kDa) were alkylated. Detection of such cysteines was carried out by Western blot analysis with anti-ATPase antibodies. It was found that shifts in the apparent molecular weight were detected only for denaturated proteins. These data suggest that available thiols are not localized on the enzyme surfaces. BN-PAGE analysis showed that the molecular weights of the ATPase complexes are almost identical in all samples. Therefore, oligomerization is probably not the reason for the inhibition of ATPase activity. Roots treated with these inhibitors in vivo exhibited stunted growth; however, a strong alkaline zone around the roots was formed only in the presence of diamide. Involvement of H+-ATPase redox regulation in this process is discussed.

Membrane physical state and stress regulation in Synechocystis: fluidizing alcohols repress fatty acid desaturation.

Cyanobacteria are prokaryotic photosynthetic organisms widely used in biotechnology, photosynthesis and abiotic stress research. There are several cyanobacterial strains modified to produce biofuels, but the influence of alcohols on cyanobacterial cell physiology is poorly understood. Here, we conducted a systematic study of the effects of nine primary aliphatic alcohols and an aromatic benzyl alcohol on both membrane physical state and the expression of genes for fatty acid desaturases (FADs) in a model cyanobacterium Synechocystis sp. strain PCC 6803. Hexan-1-ol was found to have the most membrane fluidizing action among all alcohols studied, with its efficiency correlating with both duration of treatment and alcohol concentration. A prolonged exposure to alcohol results in a continuous loss of unsaturated fatty acids (FAs) followed by cell death, an undesired challenge that should be considered in cyanobacterial biotechnology. We suggest that membrane fluidization is the key component in alcohol stress causing inactivation of FADs and resulting in a lethal depletion of unsaturated FAs. Due to the most pronounced effects of alcohol- and heat-induced membrane fluidization on desB encoding a terminal ω3-FAD, we propose to call desB a 'viscosity gene' in analogy to heat-induced 'fluidity gene' hspA.

Membrane fluidity controls redox-regulated cold stress responses in cyanobacteria.

Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.

Light-dependent cold-induced fatty acid unsaturation, changes in membrane fluidity, and alterations in gene expression in Synechocystis.

Cold stress causes unsaturation of the membrane lipids. This leads to adjustment of the membrane fluidity, which is necessary for cold acclimation of cells. Here we demonstrate that the cold-induced accumulation of PUFAs in the cyanobacterium Synechocystis is light-dependent. The desA(-)/desD(-) mutant, that lacks the genes for Δ12 and Δ6 desaturases, is still able to adjust the fluidity of its membranes in spite of its inability to synthesize PUFAs and modulate the fatty acid composition of the membrane lipids under cold stress. The expression of cold-induced genes, which are controlled by the cold sensor histidine kinase Hik33, depends on the fluidity of cell membranes and it is regulated by light, though it does not require the activity of the photosynthetic apparatus. The expression of cold-induced genes, which are not controlled by Hik33, does not depend on the membrane fluidity or light. Thus, membrane fluidity determines the temperature dependence of the expression of cold-induced genes that are under control of the Hik33, which might be the sensor of changes in the membrane fluidity. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.