Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Wielandt, Alex Green; Pedersen, Jesper Torbøl; Falhof, Janus; Kemmer, Gerdi Christine; Lund, Anette; Ekberg, Kira; Fuglsang, Anja Thoe; Pomorski, Thomas Günther; Buch-Pedersen, Morten J.; Palmgren, Michael G.

doi:10.1074/jbc.m114.617746

Cited by 35 publications

(34 citation statements)

References 56 publications

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“…The monoacyl signaling lipid lysophosphatidylcholine was the first factor found to activate the plant PM H + -ATPase (Palmgren and Sommarin, 1989). In vitro, lysophospholipids directly relieve the autoinhibitory effect of both the N-and C-terminal domains (Wielandt et al, 2015). Although lysophospholipids are the most potent activators of the PM H + -ATPase identified to date, the physiological relevance of lysophospholipids in PM H + -ATPase regulation is uncertain.…”

Section: Regulation By Lipidsmentioning

confidence: 99%

Plasma Membrane H + -ATPase Regulation in the Center of Plant Physiology

et al. 2016

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The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase.

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Section: Regulation By Lipidsmentioning

confidence: 99%

Plasma Membrane H + -ATPase Regulation in the Center of Plant Physiology

et al. 2016

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“…There are also other explanations that would account for our results. For example, it is known that detergents can activate membrane transporters (27). Thus, it is possible that detergent solubilization (the first step in obtaining purified protein after expression in yeast) converts nhTMEM16 into a partially active conformation and that this conformation is preserved even after detergent removal and reconstitution.…”

Section: Resultsmentioning

confidence: 99%

Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer

Andra

Dorsey

Royer

et al. 2018

Journal of Biological Chemistry

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Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus ) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.

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“…One can imagine that TeA binds to a site generated between the body of the protein and is stabilized by the C‐terminus. We have previously demonstrated that lysophospholipids can activate the plant PM H + ‐ATPase, whereas they do not have any effects on the fungal Pm H + ‐ATPase (Wielandt et al , ). Additionally this regulation involved the regulatory C‐terminal domain.…”

Section: Discussionmentioning

confidence: 99%

“…H + ‐ATPase activity was assayed according to the modified protocol of Baginski et al () described by Wielandt et al (). For each well, 0.5–3 μg of protein was added in a 60 μl volume.…”

Section: Methodsmentioning

confidence: 99%

Tenuazonic acid from Stemphylium loti inhibits the plant plasma membrane H⁺‐ATPase by a mechanism involving the C‐terminal regulatory domain

et al. 2020

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Summary Pathogenic fungi often target the plant plasma membrane (PM) H+‐ATPase during infection. To identify pathogenic compounds targeting plant H+‐ATPases, we screened extracts from 10 Stemphylium species for their effect on H+‐ATPase activity. We identified Stemphylium loti extracts as potential H+‐ATPase inhibitors, and through chemical separation and analysis, tenuazonic acid (TeA) as a potent H+‐ATPase inhibitor. By assaying ATP hydrolysis and H+ pumping, we confirmed TeA as a H+‐ATPase inhibitor both in vitro and in vivo. To visualize in planta inhibition of the H+‐ATPase, we treated pH‐sensing Arabidopsis thaliana seedlings with TeA and quantified apoplastic alkalization. TeA affected both ATPase hydrolysis and H+ pumping, supporting a direct effect on the H+‐ATPase. We demonstrated apoplastic alkalization of A. thaliana seedlings after short‐term TeA treatment, indicating that TeA effectively inhibits plant PM H+‐ATPase in planta. TeA‐induced inhibition was highly dependent on the regulatory C‐terminal domain of the plant H+‐ATPase. Stemphylium loti is a phytopathogenic fungus. Inhibiting the plant PM H+‐ATPase results in membrane potential depolarization and eventually necrosis. The corresponding fungal H+‐ATPase, PMA1, is less affected by TeA when comparing native preparations. Fungi are thus able to target an essential plant enzyme without causing self‐toxicity.

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Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Abstract: Background: Lysophospholipids activate P-type plasma membrane H ϩ -ATPase proton pump by an unknown mechanism.

Cited by 35 publications

References 56 publications

Plasma Membrane H + -ATPase Regulation in the Center of Plant Physiology

Plasma Membrane H + -ATPase Regulation in the Center of Plant Physiology

Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer

Tenuazonic acid from Stemphylium loti inhibits the plant plasma membrane H⁺‐ATPase by a mechanism involving the C‐terminal regulatory domain

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Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Abstract: Background: Lysophospholipids activate P-type plasma membrane H ϩ -ATPase proton pump by an unknown mechanism.

Cited by 35 publications

References 56 publications

Plasma Membrane H + -ATPase Regulation in the Center of Plant Physiology

Plasma Membrane H + -ATPase Regulation in the Center of Plant Physiology

Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer

Tenuazonic acid from Stemphylium loti inhibits the plant plasma membrane H+‐ATPase by a mechanism involving the C‐terminal regulatory domain

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Tenuazonic acid from Stemphylium loti inhibits the plant plasma membrane H⁺‐ATPase by a mechanism involving the C‐terminal regulatory domain