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Isolation of native plasma membrane H⁺-ATPase (Pma1p) in both the active and basal activation states.

ABSTRACT: The yeast plasma membrane H⁺-ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H⁺-ATPases are autoinhibited.

SUBMITTER: Pedersen JT

PROVIDER: S-EPMC5929935 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Isolation of native plasma membrane H<sup>+</sup>-ATPase (Pma1p) in both the active and basal activation states.

Pedersen Jesper Torbøl JT Kanashova Tamara T Dittmar Gunnar G Palmgren Michael M

FEBS open bio 20180325 5

The yeast plasma membrane H<sup>+</sup>-ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified p ...[more]

PMID: 29744292

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Project description:Cholesterol's effects on Na+,K+-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na+,K+-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na+,K+-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-β-cyclodextrin (mβCD). The mβCD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa3 + → E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K2 + → E1Na3 +). However, cholesterol depletion did not affect the observed rate constant for K+ occlusion by phosphorylated Na+,K+-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K2 +). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na+,K+-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na+,K+-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na+,K+-ATPase steady-state activity.

Dataset Information

Isolation of native plasma membrane H⁺-ATPase (Pma1p) in both the active and basal activation states.

Publications

Isolation of native plasma membrane H<sup>+</sup>-ATPase (Pma1p) in both the active and basal activation states.

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Dataset Information

Isolation of native plasma membrane H+-ATPase (Pma1p) in both the active and basal activation states.

Publications

Isolation of native plasma membrane H<sup>+</sup>-ATPase (Pma1p) in both the active and basal activation states.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Isolation of native plasma membrane H⁺-ATPase (Pma1p) in both the active and basal activation states.