ACIDIFICATION OF THE YOUNG PHAGOSOMES OF PARAMECIUM IS MEDIATED BY PROTON PUMPS DERIVED FROM THE ACIDOSOMES

Although it is generally accepted that phagosome acidification is indu ced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to th e phagosomes is not well understood. To study this question in Paramec ium, it was necessary to first show that an authentic V-ATPase was pre sent on their phagosomal membranes. Three antibodies raised against V- ATPases or their subunits were each found to label one or two large di gestive vacuoles (DVs) in Paramecium multimicronucleatum when immunofl uorescence microscopy was used. Using horseradish peroxidase immunocyt ochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting t he vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamy cin B, which at 10 nM doubled the tin for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs of Paramecium have a bon a-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V -ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have n ow determined that Paramecium has two immunologically related V-ATPase s that are involved in two very different functions, (1) the acidifica tion of phagosomes and (2) fluid segregation in the contractile vacuol e complexes.