Proton transport by a fraction of endoplasmic reticulum and Golgi membranes of corn roots: comparison with the plasma membrane and tonoplast H + pumps

Abstract

Microsomal membranes isolated from roots of Zea mays, L. were separated into three fractions (F16, F34 and F40) by sucrose gradient centrifugation. The low density membranes (F16), which were previously shown to be enriched in marker enzymes characteristic of endoplasmic reticulum (ER) and Golgi membranes, have a proton pumping system which was compared to that of the tonoplast (F34) and of the plasma membranes (F40). We observed that ER-Golgi membranes have an ATPase activity (∼100 nmol Pi/mg protein/min) which, under certain conditions, is stimulated by sulfate and it is neither sensitive to vanadate nor to nitrate. This ATPase activity generates a transmembrane proton gradient (ΔpH), which is not affected by vanadate, but it is strongly inhibited by sulfate and nitrate. In contrast, nitrate greatly stimulates, whereas vanadate strongly inhibits the H + pumping by the plasma membrane vesicles (F40). Chloride improves ΔpH generation in the three fractions studied, but only the F16 and F34 H +-ATPase appear to be directly affected by this anion. Regarding the effect of cations, we observed that K +, in all types of membranes, is not required for the operation of the H + pump activity, although the plasma membrane H + pump appears to be slightly stimulated by K +. In contrast, Na + did not alter the H + accumulation in the three membrane fractions tested. We also observed that the H + pumping of the ER-Golgi membranes is distinguishable from the others by its high sensitivity to the reagent for sulphydryl groups, N-ethylmaleimide (NEM), whereas the plasma membrane H + pump is distinguished by its sensitivity to the reagent for amino groups, trinitrobenzenesulfonate (TNBS) and to an alkaline pH (7.7). The results indicate that ER-Golgi membranes contain a (V type)-H + pump, which is distinct from the (V type)-tonoplast H + pump and from the (P type)-plasma membrane H + pump.