INVOLVEMENT OF THE VACUOLAR PROTON-TRANSLOCATING ATPASE IN MULTIPLE STEPS OF THE ENDO-LYSOSOMAL SYSTEM AND IN THE CONTRACTILE VACUOLE SYSTEM OF DICTYOSTELIUM-DISCOIDEUM

We have investigated the effects of Concanamycin A (CMA), a specific i nhibitor of vacuolar type H+-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoid eum. This drug inhibited acidification and increased the pH of endolys osomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, an d phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al, (1989) J. Biol. Chem. 264, 3454-3463) tha t maintenance of acidic pH in lumenal compartments is required for eff icient processing and targeting of a lysosomal enzyme, alpha-mannosida se. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presen ce of CMA, swelled rapidly and ruptured. Fluorescence microscopy revea led that CMA treatment induced gross morphological changes in D. disco ideum cells, characterized by the formation of large intracellular vac uoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA af ter one hour of drug treatment even in the absence of protein synthesi s. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappe ared, endo-lysosomal pH decreased, and the rate of endocytosis, exocyt osis and phagocytosis returned to normal. This study demonstrates that the V-H+-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoide um can compensate for the loss of a functional V-H+-ATPase.