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

• Published in: Science (American Association for the Advancement of Science) Vol. 351; no. 6280; pp. 1469 - 1473
• Main Authors: Veshaguri, Salome, Christensen, Sune M., Kemmer, Gerdi C., Ghale, Garima, Møller, Mads P., Lohr, Christina, Christensen, Andreas L., Justesen, Bo H., Jørgensen, Ida L., Schiller, Jürgen, Hatzakis, Nikos S., Grabe, Michael, Pomorski, Thomas Günther, Stamou, Dimitrios
• Format: Journal Article
• Language: English
• Published: United States American Association for the Advancement of Science 25.03.2016; The American Association for the Advancement of Science
• Subjects: Allosteric Regulation; Arabidopsis Proteins - antagonists & inhibitors; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - metabolism; Biochemistry; Enzymes; Eukaryotes; Hydrogen-Ion Concentration; Ion Transport; Membrane Potentials - drug effects; Membrane Potentials - physiology; Molecular Imaging; Protein Structure, Tertiary; Proton-Translocating ATPases - antagonists & inhibitors; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - metabolism; Protons; Valinomycin - pharmacology
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.aad6429

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.