Abstract
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.
Highlights
P-type H+ -ATPases of plant cells transform the energy of ATP hydrolysis into a transmembrane electrochemical proton gradient (∆μH+ ), in which dissipation establishes the transfer of ions and metabolites across the plasma membrane [1,2,3]
Plasmalemma obtained through the partitioning of microsomal membranes in an aqueous two-phase polymer system contains all of the main classes of membrane lipids: phospholipids (1–3, 5), cerebrosides (6), and sterols (including steryl glycosides and acyl steryl glycosides (7, 8)) as well as minor amounts of phosphatidic acid (4) and free fatty acids (9) (Figure 1a)
The chromatogram illustrates that the ratio between phospholipids and sterols in the plasma membrane is close to 1, as was shown in [44]
Summary
P-type H+ -ATPases of plant cells transform the energy of ATP hydrolysis into a transmembrane electrochemical proton gradient (∆μH+ ), in which dissipation (along with corresponding transporters) establishes the transfer of ions and metabolites across the plasma membrane [1,2,3]. The plasma membrane H+ -ATPase of plant cells is characterized by the presence of the so-called “activated state”, which occurs when autoinhibition is lost and the C-terminal domain of the protein is involved [8,9] This state is distinguished by a change in the. The kinetic parameters of the enzyme can vary and can be characterized by an increase in Vmax and affinity for ATP as well as a shift in the optimal pH of the enzyme to the more alkaline region, indicating an increased affinity for protons Such effects are most clearly manifested in the presence of fusicoccin, which binds adaptor 14-3-3 proteins and the phosphorylated C-terminal domain of the ATPase [12,13]
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