Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector [Membrane Biology]

September 28th, 2015 by Couoh-Cardel, S., Milgrom, E., Wilkens, S.

The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity and its structure was characterized by electron microscopy (EM) of single molecules and two-dimensional (2-D) crystals. Projection images of negatively stained Vo 2-D crystals showed a ring like structure with a large asymmetric mass at the periphery of the ring. A cryo EM reconstruction of Vo from single particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of 3-D reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1 and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~ 5 μM. Treatment of purified Vo sector with lyso 1-palmitoyl phosphatidylglycerol (LPPG) resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.