Direct observation of proton pumping by a eukaryotic P-type ATPase

Abstract

In eukaryotes, P-type ATPases generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements combined with a physical non-equilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 s) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states, but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates, but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters.

Dynamics of P-type ATPase transport revealed by single-molecule FRET

“…Such investigations will be greatly aided by imaging transport from multiple structural perspectives to resolve how the motions of each individual cytoplasmic domain couple to the transmembrane bundle. A powerful extension would be simultaneous measurements of substrate transport tracked also at a single-molecule scale 42 . Such pursuits will be greatly aided by the continued development of chemical strategies enabling the site-specific labeling of native, cysteine-containing integral membrane proteins as well as robust strategies for incorporating ligand-specific fluorescence sensors within the lumen of reconstituted proteoliposomes 43 .…”

Improved Model of Proton Pump Crystal Structure Obtained by Interactive Molecular Dynamics Flexible Fitting Expands the Mechanistic Model for Proton Translocation in P-Type ATPases

“…Interestingly, single-molecule studies of AHA2 function have been introduced and suggest also that inactive states and protein-mediated proton leaks must be considered as part of the functional cycle (Veshaguri et al, 2016), thus many structural and mechanistic questions remain to be addressed to get a full insight into the inner workings of the plasma-membrane proton pump.…”

Size-dependent, stochastic nature of lipid exchange between nano-vesicles and model membranes

“…The aim of the work is to decipher the dominant mechanism for the exchange, being either transfer of individual lipids in many steps or the transfer of many lipids in one or a few steps as an effect of stalk formation. To overcome these limitations, a range of assays, based on microscopy imaging of surface adhering vesicles, have been developed for a range of studies, including vesicles fusion, 25 membrane transport, 26,27 protein/peptidemembrane interaction [28][29][30] and membrane active enzymes. The close contact between membranes can be established by nonspecific interactions, 19 such as electrostatic charge on membrane surfaces, [20][21][22][23] or forces due to solute depletion in the inter-membrane hydration layer.…”