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.
Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e.g., fluorescent tags. Preparative separation of membrane protein loaded nanodiscs from empty nanodiscs and protein aggregates results in monodisperse nanodisc preparations ideal for structural and functional characterization using biophysical methods.
Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra-and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.
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