Figure 5.

Reconstitution of concanamycin-sensitive V-ATPase complexes in the presence and absence of RAVE.A, RAVE enhances V-ATPase reassembly and acquisition of concanamycin A–sensitive activity. Activity for two replicates from each of two independent experiments (using different preparations of components) is plotted for two time points of incubation. Red columns, no RAVE added; blue columns, RAVE added. Error bars represent SEM. B, time course of V-ATPase assembly. V₁-H_chim, Vph1–V_o nanodiscs and expressed subunit C were combined in a 1:1:3 M ratio and incubated at room temperature for the indicated times. Samples with RAVE had an equimolar concentration of RAVE, for a final of 210 nM each of RAVE, V₁-H_chim and V_o, and 630 nM subunit C, specific activity represents the ATPase activity sensitive to 200 nM concanamycin A. Each point represents the average of two replicates. Fits of each curve to a single exponential equation are indicated. RAVE, Regulator of the H⁺-ATPase of Vacuoles and Endosomes; V-ATPase, vacuolar H⁺-ATPase.