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.

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the β2-adrenergic receptor using ~109-fold less protein than conventional assays.

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