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

Science  25 Mar 2016:
Vol. 351, Issue 6280, pp. 1469-1473
DOI: 10.1126/science.aad6429

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A proton pump in action

P-type adenosine triphosphatases (ATPases) use the energy from ATP hydrolysis to pump cations across biological membranes. The electrochemical gradients that are generated control many essential cellular processes. Veshaguri et al. incorporated a plant proton pump into vesicles and monitored the dynamics of single pumps. Pumping was stochastically interrupted by long-lived inactive or leaky states. The work reveals how these proton pumps are regulated by a protein domain and by pH gradients.

Science, this issue p. 1469

Abstract

In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. 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 nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) 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.

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