Bafilomycin A1 Prevents Maturation of Autophagic Vacuoles by Inhibiting Fusion between Autophagosomes and Lysosomes in Rat Hepatoma Cell Line, H-4-II-E Cells.

Abstract

ABSTRACT. Westudied the effects of bafilomycin Ai, a potent and specific inhibitor of vacuolar 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 Ai 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 Ai, suggesting that fusion between autophagosomes and lysosomes was disturbed by this drug. Inhibition of the fusion was reversible, and the autophagosomes changed into autolysosomes after the removal of the inhibitor. Bafilomycin Ai also prevented the appearance of endocytosed HRPin autophagic vacuoles. These results suggested that acidification of the lumenal space of autophagosomesor lysosomes by V-ATPase is important for the fusion between autophagosomes and lysosomes.Autophagy is one of the main pathways of the degradation of the endogenous proteins and organella (4, ll, 32). In the process of autophagy, membrane structures called isolation membranesor phagophores appear, segregate parts of the cytoplasm, and form autophagosomes. The newly formed autophagosomes (early autophagosomes) fuse with endosomes or prelysosomes, and becomea more advanced stage of autophagosomes (late autophagosomes or amphisomes) of acidic luminal pH (2, 1 1, 12). The autophagosomes then acquire hydrolytic enzymes by fusion with lysosomes, and are transformed into mature autolysosomes in which degradation of the content proceeds (10, ll, 40). Vacuolar H+ ATPase (V-ATPase) is localized in organelles of the central vacuolar system such as coated vesicles, endosomes, lysosomes, chromaffin granules, and Golgi apparatus, and plays an important role by maintaining the acidic environment in these compartments (for review, see Mellman et al). Bafilomycin Ai, a macrolide antibiotic isolated from Streptomyces sp., is a highly specific inhibitor of the V-ATPase (5, 14, 38). Inhibition by bafilomycin Ai occurs through binding to the membrane-spanning pore forming domain of the enzyme (7, 42). Bafilomycin Ai was also effective on living cells when added extracellularly (36, 41). Therefore it is a powerful tool to study the role of V-ATPase and vacuolar pH. Using this drug, we have previously reported that V-ATPase is essential for acidifying the lumen of lysosomes and subsequent protein degradation of endocytosed epidermal growth factor (EGF) in lysosomes of cultured cells (41) and phagocytosed rod outer segmentsin phagolysosomesof retinal pigmentepithelial cells (9).In this study, to clarify the roles of V-ATPasein autophagy, we studied the effects of bafilomycin Ax on the process of autophagy in rat hepatoma cell line, H-

Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates

“…To further evaluate the autophagic flux, we assessed the levels of LC3-II and p62 in the presence of bafilomycin A1 (Baf A1), a proton pump inhibitor that blocks the lysosomal acidification and the AP-lysosome fusion. 10 In the presence of Baf A1, the PLD1 inhibitor did not alter the level of LC3-II or p62 (Figure 1c), indicating that the increases in these proteins upon PLD1 inhibition were due to the impaired autophagic flux. We next measured the fusion rate of APs to lysosomes by using LC3 tagged with GFP-RFP tandem fluorescent proteins (tfLC3).…”

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

“…Bafilomycin A1 is a specific inhibitor of the lysosomal protein pump (vacuolar H þ -ATPase). 21,22 ATP is required at all steps of autophagy. 21 In agreement with this finding, bafilomycin A1 suppresses autophagy by inhibiting fusion between autophagosomes and lysosomes.…”

“…21,22 ATP is required at all steps of autophagy. 21 In agreement with this finding, bafilomycin A1 suppresses autophagy by inhibiting fusion between autophagosomes and lysosomes. This inhibitory effect may be due to a requirement that the lysosome be acidified for fusion with the autophagosome.…”

Programmed cell death 50 (and beyond)

“…In this case, autophagy continues until mitochondria are destroyed, thus depriving the cells of any possibility of recovery. [47][48][49] The genetic analysis of autophagy in yeast, primarily by Klionsky and others, 50,51 the extension of the genetics to mammalian cells, primarily by Levine,[52][53][54] and the development of new tools to study lysosomes, including fluorescent markers 55 and lysosome-specific drugs 56,57 have clarified the situation considerably and allowed much closer examination of what autophagy does. It now appears that autophagy usually protects cells 5,58 in that cells that can activate autophagy withstand many types of stresses far better than cells that cannot.…”