THE PEGS ON THE DECORATED TUBULES OF THE CONTRACTILE VACUOLE COMPLEX OF PARAMECIUM ARE PROTON PUMPS

Citation
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
Citations number
22
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
00219533
Volume
108
Year of publication
1995
Part
10
Pages
3163 - 3170
Database
ISI
SICI code
0021-9533(1995)108:<3163:TPOTDT>2.0.ZU;2-U
Abstract
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.