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

Abstract

The yeast plasma membrane H + ‐ ATP ase Pma1p is a P‐type ATP ase 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 + ‐ ATP ases are autoinhibited.

The Oligomeric State of the Plasma Membrane H+-ATPase from Kluyveromyces lactis

“…Further, the calculated AMP-PCP affinity agree with that reported in the recombinant H + -ATPase N-domain from K. lactis [22]. The calculated binding affinities for nucleotides indicates that the aggregated hexamer (Peak 1) is the activated state of H + -ATPase, while the free hexamer probably represents the basal active state as proposed recently [46].…”

“…Therefore, the two isolated hexameric populations (aggregated and free) probably represent the activated and basal state of the H + -ATPase, respectively. In agreement with this proposal, it has been reported recently that two H + -ATPase hexameric populations coexist in plasma membranes [ 46 ]. H + -ATPase self-segregation in plasma membranes seems to be dependent on the interactions of certain phospholipids and cholesterol with the TM α-helices [ 16 , 17 , 18 , 30 , 51 , 52 ].…”

Energy costs of salt tolerance in crop plants

“…Just the stoichiometry of the proton pump could have a profound effect on the energetics, and an H + : ATP < 1 (e.g. when the pump is not activated (Pedersen et al ., )) would reduce the energy efficiency substantially; it is interesting that cytosolic K + and potentially Na + can have an effect on this (Buch‐Pedersen et al ., ). The identification of molecular components of energetically costly processes can be used to engineer plants for improved yield (Amthor et al ., ).…”

Promotion and Upregulation of a Plasma Membrane Proton-ATPase Strategy: Principles and Applications

“…PM H + -ATPase is a member of the P-type ATPase super family and is the primary active transport system in plants and fungi ( Sussman, 1994 ). The activated state of PM H + -ATPase results in an electrochemical gradient that is the driving force behind the transport of other solutes across the cell membrane ( Palmgren, 2001 ; Pedersen et al, 2018 ). This process involves the hyperpolarization of the plasma membrane and the hydrolysis of ATP, and these two steps occur almost simultaneously ( Morsomme and Boutry, 2000 ).…”