Bafilomycin A₁ prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells

We studied the effects of bafilomycin A₁, a potent and specific inhibitor of vaculoar H⁺ ATPase (V-ATPase), on the process of autophagy in rat hepatoma cell line, H-4-II-E cells. To induce autophagy, cells were transferred from Dulbecco's modified Eagle medium containing 12% fetal calf serum into Hanks' balanced salt solution. When bafilomycin A₁ was added to Hanks' balanced salt solution endogenous protein degradation was strongly inhibited and numerous autophagosomes accumulated in H-4-II-E cells, whereas autolysosomes decreased in number. Acid phosphatase activity was not detected in the autophagosomes which accumulated in the presence of bafilomycin A₁, suggesting that fusion between autophagosomes and lysosomes was disturbed by this drub. Inhibition of the fusion was reversible, and the autophagosomes changed into autolysosomes after the removal of the inhibitor. Bafilomycin A₁ also prevented the appearance of endocytosed HRP in autophagic vacuoles. These results suggested that acidification of the lumenal space of autophagosomes or lysosomes by V-ATPase is important for the fusion between autophagosomes and lysosomes.