INVOLVEMENT OF THE VACUOLAR PROTON-TRANSLOCATING ATPASE IN MULTIPLE STEPS OF THE ENDO-LYSOSOMAL SYSTEM AND IN THE CONTRACTILE VACUOLE SYSTEM OF DICTYOSTELIUM-DISCOIDEUM
La. Temesvari et al., INVOLVEMENT OF THE VACUOLAR PROTON-TRANSLOCATING ATPASE IN MULTIPLE STEPS OF THE ENDO-LYSOSOMAL SYSTEM AND IN THE CONTRACTILE VACUOLE SYSTEM OF DICTYOSTELIUM-DISCOIDEUM, Journal of Cell Science, 109, 1996, pp. 1479-1495
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.