Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H + pump.This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H + pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H ÷ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump.These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.Many intracellular organelles are membrane bounded, a characteristic that allows the development ofa microenvironment difference in composition is usually achieved by ion transport processes that are energized by cellular metabolism. In a number of cells and organeiles including procaryotes, mitochondria, lysosomes, and endocytic vesicles, proton electrochemical gradients generated by H ÷ pumps play an important role in signal transduction and energy conversion. We were stimulated by the recent discovery that monensin, an ionophore that exchanges H + for sodium (or potassium), had dramatic effects on the structure and function of the Golgi apparatus (1, 2). Since the addition of monensin collapses a pH gradient, which could be the result of a H ÷ pump, we isolated Golgi vesicles and tested them for the presence of a proton pump. We found that a fraction of rat liver membranes, highly enriched in a Golgi marker, contained an electrogenic ATP-dependent proton pump. These membranes also appeared to contain a chloride conductance in parallel with this pump. MATERIALS AND METHODSOolgi membranes were prepared according to the procedure of Wibo et al. (3). Livers of starved rats were excised, weighed, finely minced with a razor blade, and then homogenized in 0.5 M sucrose with 20 strokes in a Dounce-type homogenizer fitted with a loose pestle in 2-3 ml of homogenizing buffer per gram tissue. Homogenizing buffer was 0.5 M sucrose which contained (as did all other sucrose solutions) 37.5 mM Tris-maleate, pH 6.5, 1% Dextran T-500 (Pharmacia, Inc., Piscataway, N J), 5 mM MgCl2, and l mM dithiothreitol, unless otherwise specified. The homogenate was centrifuged for 10 rain at 2,100 rpm in a Sorvall centrifuge with the SA-600 rotor (DuPont Co., Instruments Products, Newtown, CT). The supernatant was decanted and the pellet