Ak. Fok et al., THE PEGS ON THE DECORATED TUBULES OF THE CONTRACTILE VACUOLE COMPLEX OF PARAMECIUM ARE PROTON PUMPS, Journal of Cell Science, 108, 1995, pp. 3163-3170
Our previous study has shown that the decorated tubules (collectively
known as the decorated spongiome) of the contractile vacuole complex (
CVC) in Paramecium are the site of fluid segregation, as the binding o
f microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced
the CVC's fluid output, In this study, we showed by immunogold labelin
g on cryosections that the antigenic sites for mAb DS-1 were located o
n the 15 nm 'pegs' protruding from the cytosolic surface of the decora
ted tubules, In immunofluorescence studies, both polyclonal antibodies
against the subunits of the V-ATPase of Dictyostelium discoideum and
against the 57 kDa B-subunit of the V-ATPase of chromaffin granules ga
ve identical labeling patterns to that produced by mAb DS-1, On cryose
ctions, all three antigens were located most consistently near or on t
he pegs of the decorated tubules, These data support the notion that t
he pegs on the membrane of the decorated tubules represent the V-1 com
plex of a proton pump, Concanamycin B, a potent inhibitor of V-ATPase
activity and of acidification of lysosomes and endosomes, strongly and
reversibly inhibited fluid output from the CVC but had minimal effect
on the integrity of the decorated spongiome as observed by immunofluo
rescence. Such inhibition suggests that a V-ATPase is intimately invol
ved in fluid segregation, Exposing Paramecium to 12 degrees C or 1 deg
rees C for 30 minutes resulted in the dissociation of the decorated tu
bules from the smooth spongiome that borders the collecting canals; th
us the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were a
ll found dispersed in the cytosol, Fluid output in these cells also ce
ased, In summary, the results obtained from proton pump antibodies, a
V-ATPase inhibitor and cold treatment showed that the pegs on the deco
rated spongiome of Paramecium are V-1 subunits of the V-ATPase and tha
t these V1V0 complexes must remain active and in place for normal flui
d segregation to occur.