Abstract
In all eukaryotes the plasma membrane potential and secondary transport systems are energized
by P-type ATPases whose regulation however 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 nonequilibrium
model of vesicle acidification, revealed that pumping is stochastically interrupted by
long-lived (~100 s) inactive or leaky states. The autoinhibitory regulatory R-domain of AHA2
reduced the intrinsic pumping rates but counter-intuitively increased the time spent pumping.
Allosteric regulation by pH gradients affected the time spent pumping and the leakage
probability but surprisingly not the intrinsic pumping rate. Interestingly, ATP dilution decreased
the ATP hydrolysis rates in bulk while single molecule data revealed that intrinsic pumping rates
remained constant. Titration of ATP down to ~1% of apparent Km for ATPase activity
exclusively affected the distributions of the durations the pump spends in active and inactive
states. The dramatic consequence of our findings is that ATP reduction decreased ATP/H+
stoichiometry of the pump. We propose that variable ATP/H+ stoichiometry emerges as a novel
mechanism for adaptation when challenged with depletion of ATP that is likely relevant for other
ATPases. Such measurements will provide indispensable insights into the mechanisms of
function and regulation of many other ion-coupled transporters.
by P-type ATPases whose regulation however 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 nonequilibrium
model of vesicle acidification, revealed that pumping is stochastically interrupted by
long-lived (~100 s) inactive or leaky states. The autoinhibitory regulatory R-domain of AHA2
reduced the intrinsic pumping rates but counter-intuitively increased the time spent pumping.
Allosteric regulation by pH gradients affected the time spent pumping and the leakage
probability but surprisingly not the intrinsic pumping rate. Interestingly, ATP dilution decreased
the ATP hydrolysis rates in bulk while single molecule data revealed that intrinsic pumping rates
remained constant. Titration of ATP down to ~1% of apparent Km for ATPase activity
exclusively affected the distributions of the durations the pump spends in active and inactive
states. The dramatic consequence of our findings is that ATP reduction decreased ATP/H+
stoichiometry of the pump. We propose that variable ATP/H+ stoichiometry emerges as a novel
mechanism for adaptation when challenged with depletion of ATP that is likely relevant for other
ATPases. Such measurements will provide indispensable insights into the mechanisms of
function and regulation of many other ion-coupled transporters.
| Original language | English |
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| Publisher | Department of Chemistry, Faculty of Science, University of Copenhagen |
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| Number of pages | 144 |
| Publication status | Published - 2015 |