Purification and properties of a cytosolic V-1-ATPase

The native V-1 complex of the tobacco hornworm vacuolar type ATPase (V-ATPase) was purified from cytosolic extracts of molting larval midgut. It consisted of the established V-ATPase subunits A, B, and E along with the 14-kDa subunit F and the novel 13-kDa subunit G, The final amount of purified V-1 complex made up an unexpectedly high 2% of the total cytosolic protein, with a yield of similar to 0.4 mg/g of tissue. An equally high amount of cytosolic V-1 complex was obtained from starving intermolt larvae, By contrast, the cytosolic V-1 pool was reduced drastically in feeding intermolt larvae or in larvae that had been refed after starvation. The activity of the membrane-bound V-ATPase holoenzyme was inversely related to the size of the cytosolic V-1 pool, suggesting that the insect plasma membrane V-ATPase is regulated by reversible disassembly of the V-1 complex as a function of the feeding condition of the larvae. Like F-1-ATPases, the purified V-1 complex exhibited Ca2+-dependent ATPase activity and, in the presence of 25% methanol, exhibited Mg2+-dependent ATPase activity. Therefore, we designate the native V-1 complex, V-1-ATPase. Both enzyme activities were completely inhibited by micromolar-N-ethylmaleimide. In contrast to the Ca2+-dependent V-1-ATPase activity, the Mg2+/methanol-dependent V-1-ATPase activity did not decrease with the incubation time and thus was not inhibited by ADP. Methanol appears to induce a conformational change of the V-1 complex, leading to enzymatic properties of the V-1-ATPase that are similar to those of the membrane-bound V-ATPase holoenzyme. This is the first time that a native and enzymatically active V-1 complex has been purified from the cytosol.