AN IMMUNOCYTOCHEMICAL ANALYSIS OF THE VACUOLAR PROTON PUMP IN DICTYOSTELIUM-DISCOIDEUM

Antisera were generated in rabbits against the vacuolar proton pump (V -H+-ATPase) purified from Dictyostelium discoideum. The antisera inhib ited V-H+-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H+-ATPase from c ell homogenates. Immunocytochemical analysis of intact cells and subce llular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shape s, which corresponded to the acidosomes. The cytoplasmic surfaces of l ysosomes, phagosomes and the tubular spongiome of the contractile vacu ole also bore the pump antigen. The lumina of multivesicular bodies we re often stained intensely; the internalized antigen may have been der ived from acidosomes by autophagy. Antibodies against V-H+-ATPases fro m plant and animal cells cross-reacted with the proton pumps of Dictyo stelium. Antisera directed against the V-H+-ATPase of Dictyostelium de corated a profusion of small vacuoles scattered throughout the cytopla sm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. W e conclude that the V-H+-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.