Kv. Nolta et al., AN IMMUNOCYTOCHEMICAL ANALYSIS OF THE VACUOLAR PROTON PUMP IN DICTYOSTELIUM-DISCOIDEUM, Journal of Cell Science, 105, 1993, pp. 849-859
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.