Purification and Properties of a Cytosolic V1-ATPase

Ralph Gräf,William R Harvey,Helmut Wieczorek

doi:10.1074/jbc.271.34.20908

Ralph Gräf, William R Harvey + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.34.20908

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Abstract

The native V1 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 V1 complex made up an unexpectedly high 2% of the total cytosolic protein, with a yield of approximately 0.4 mg/g of tissue. An equally high amount of cytosolic V1 complex was obtained from starving intermolt larvae. By contrast, the cytosolic V1 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 V1 pool, suggesting that the insect plasma membrane V-ATPase is regulated by reversible disassembly of the V1 complex as a function of the feeding condition of the larvae. Like F1-ATPases, the purified V1 complex exhibited Ca2+-dependent ATPase activity and, in the presence of 25% methanol, exhibited Mg2+-dependent ATPase activity. Therefore, we designate the native V1 complex, V1-ATPase. Both enzyme activities were completely inhibited by micromolar N-ethylmaleimide. In contrast to the Ca2+-dependent V1-ATPase activity, the Mg2+/methanol-dependent V1-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 V1 complex, leading to enzymatic properties of the V1-ATPase that are similar to those of the membrane-bound V-ATPase holoenzyme. This is the first time that a native and enzymatically active V1 complex has been purified from the cytosol.

Highlights

The final amount of purified V1 complex made up an unexpectedly high 2% of the total cytosolic protein, with a yield of ϳ0.4 mg/g of tissue
Purification of the Cytosolic V1 Complex from Molting Larvae—Sumner et al [22] recently demonstrated that V1 subunits were removed from the membrane during molt. It was not clear whether the V1 complex remained complete or whether the V1 subunits disintegrated, there was some evidence for the first alternative since cytosolic V1 pools had already been described in yeast and in a kidney cell line [20, 21]
The V1 complex was composed of the established subunits A, B, and E along with the 14-kDa subunit F [8] and the novel 13-kDa subunit G [9]

Summary

The abbreviations used are

V-ATPase, vacuolar type ATPase; FPLC, fast protein liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; GCAM, goblet cell apical membranes; CF1, coupling factor 1. Regulated during the molt by detachment of V1 subunits from the membrane. The tobacco hornworm V-ATPase may serve as a valuable model for V-ATPases in general since it is well characterized [23] and all five of its known V1 subunits have been cloned and sequenced (8, 9, 24 –26). We show that the midgut cytosolic extract from molting tobacco hornworms is a rich source for the purification of enzymatically active V1 complexes. We present evidence that the size of the cytosolic V1 pool depends on the feeding condition of the M. sexta larvae.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION