A partially purified H+-ATPase from the plasma membrane (PM) of corn (Zea mays L.) roots was inserted into vesicles prepared with soybean (Glycine max 1.) phospholipids and various concentrations of individual sterols using either a freeze-thaw sonication or an octylglucoside dilution procedure. Both methods yielded a functional enzyme that retained i t s native characteristics. We have investigated the effects o f typical plant sterols (i.e. sitosterol, stigmasterol, and 24-methylcholesterol) on both ATP hydrolysis and H + pumping by the reconstituted corn root PM ATPase. We have also checked the influence of cholesterol and of two unusual sterols, 24-methylpollinastanol and 14a,24-dimethylcholest-8-en-3~-ol. Here we present evidence for a sterol modulation of the plant PM H+-ATPase activity. I n particular, cholesterol and stigmasterol were found t o stimulate the pump, especially when present at 5 mol%, whereas all of the other sterols tested behaved as inhibitors at any concentration in proteoliposomes. In all situations H + pumping was shown t o be more sensitive t o a sterol environment than was ATP hydrolysis. Our results suggest the occurrence of binding sites for sterols on the plant PM H+-ATPase.The PM H+-ATPase from higher plant cells is a P-type ion-translocating ATPase that produces an inwardly directed proton electrochemical gradient across the PM, thereby providing the driving force for secondary transport of nutrients such as anions, amino acids, sugars, or hormones into the cells (Serrano, 1989; Michelet and Boutry, 1995). The activity of the PM H'-ATPase also contributes to the maintenance of intracellular pH, and regulation of this process has been proposed to mediate a broad range of cellular processes involved in the growth, development, and response of plants to environmental and hormonal signals (Marré and Ballarin-Denti, 1985;Serrano, 1989; Michelet et Boutry, 1995). It is now well established that this enzyme is very sensitive to changes in its lipid environment, and recently much work has been devoted to the requirement of the enzyme for various kinds of lipids (Cooke and Burden, 1990;Palmgren, 1991; Kasamo and Sakakibara, 1995 has been paid to sterols, which are the other major components of the PM (Hartmann and Benveniste, 1987) and might also participate in the regulation of the PM HtATPase, as cholesterol does in the case of the Na+,K+-ATPase (Yeagle et al., 1988; Cornelius, 1995) or the sarcoplasmic reticulum Ca*+-ATPase (Simmonds et al., 1982; Ding et al., 1994).Plant cells contain a mixture of sterols (sitosterol, stigmasterol, and 24-methylcholesterol) instead of the one major sterol-cholesterol or ergosterol-found in mammalian or funga1 cells, respectively. In the present study we have investigated the effects of various individual sterols on ATP hydrolysis and ATP-driven H+ translocation by a partially purified PM H+-ATPase reconstituted into soybean (Glycine max L.) phospholipid vesicles by means of two different methods. We have examined the effects of the typical pla...