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Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H⁺-ATPase.

ABSTRACT: The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H⁺-ATPase Arabidopsis thaliana H⁺-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H⁺-ATPase activity.

SUBMITTER: Paweletz LC

PROVIDER: S-EPMC10488199 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase.

Paweletz Laura C LC Holtbrügge Simon L SL Löb Malina M De Vecchis Dario D Schäfer Lars V LV Günther Pomorski Thomas T Justesen Bo Højen BH

International journal of molecular sciences 20230823 17

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytopl ...[more]

PMID: 37685912

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Project description:Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8-10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8-10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.

Dataset Information

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H⁺-ATPase.

Publications

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H<sup>+</sup>-ATPase.

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