title: Direct observation of proton pumping by a eukaryotic P-type ATPase.
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.
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Salome
authors.initials: S
authors.lastname: Veshaguri
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Sune M
authors.initials: SM
authors.lastname: Christensen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg Denmark.
authors.forename: Gerdi C
authors.initials: GC
authors.lastname: Kemmer
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Garima
authors.initials: G
authors.lastname: Ghale
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Mads P
authors.initials: MP
authors.lastname: Møller
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christina
authors.initials: C
authors.lastname: Lohr
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Andreas L
authors.initials: AL
authors.lastname: Christensen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg Denmark.
authors.forename: Bo H
authors.initials: BH
authors.lastname: Justesen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg Denmark.
authors.forename: Ida L
authors.initials: IL
authors.lastname: Jørgensen
authors.affiliation: Institute of Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Leipzig, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schiller
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Nikos S
authors.initials: NS
authors.lastname: Hatzakis
authors.affiliation: Cardiovascular Research Institute, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Grabe
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg Denmark.
authors.forename: Thomas Günther
authors.initials: TG
authors.lastname: Pomorski
authors.affiliation: Bionanotecnology and Nanomedicine Laboratory, University of Copenhagen, Copenhagen, Denmark. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Nano-Science Center, University of Copenhagen, Copenhagen, Denmark. Lundbeck Foundation Center Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Stamou
mesh_terms: D000494:Allosteric Regulation; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D057054:Molecular Imaging; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D029681:Arabidopsis Proteins; D011522:Protons; D014634:Valinomycin; D006180:Proton-Translocating ATPases; C479903:AHA2 protein, Arabidopsis