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-

No abstract

No abstract

We have previously identified two species of the low-affinity human Fc receptor for IgE, FceRIIa and FceRIIb, which differ only in a short stretch of amino acids at the N-terminal cytoplasmic end. (1)(2)(3). FceRII has been known as a B-cell differentiation antigen, CD23 (4-7), and has been proposed to be involved in several B-cell functions such as the growthpromoting effect (8, 9), cell adhesion (10), and IgE-mediated antigen presentation.(11). On the other hand, FceRII has also been reported to be expressed on macrophages, eosinophils, and platelets and to be an effector molecule of IgE-mediated immunity, including immediate-type allergy and cytotoxicity against certain parasites (2). IgE can mediate release of varieties of chemical mediators from eosinophils, monocytes, and platelets (12)(13)(14).We have previously identified two species of human FceRII, FceRIIa and FceRIIb (15, 16). They are generated by utilizing different transcriptional initiation sites and 5' exons of the single genomic gene, and they differed only in a short amino acid stretch in the N-terminal cytoplasmic end. FceRIIa is constitutively, but cell-type-specifically, expressed on B cells. On the other hand, monocytes and eosinophils express only FceRIlb. FceRIIb is expressed on normal peripheral blood B cells and monocytes only after stimulation with interleukin 4, which is known to be the cytokine responsible for the isotype switching of B cells to 5030The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ABSTRACT. Ultrastructural localization of I11SP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. Wenoticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. Wesuggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like I11SP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.Inositol