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-

Hrs is a 115-kDa double zinc finger protein that is rapidly tyrosine phosphorylated in growth factor-stimulated cells. However, its function remains unknown. Here we show that Hrs is localized to early endosomes. Intracellular localization of endogenous Hrs and exogenously expressed Hrs tagged with the hemagglutinin epitope was examined by immunofluorescence staining using anti-Hrs and anti-hemagglutinin epitope antibodies, respectively. Hrs was detected in vesicular structures and was colocalized with the transferrin receptor, a marker for early endosomes, but only partially with CD63, a marker for late endosomes. A zinc finger domain deletion mutant of Hrs was also colocalized with the transferrin receptor, suggesting that the zinc finger domain is not required for its correct localization. Immunoelectron microscopy showed that Hrs was localized to the cytoplasmic surface of these structures. By subcellular fractionation, Hrs was recovered both in the cytoplasmic and membrane fractions. The membrane-associated Hrs was extracted from the membrane by alkali treatment, suggesting that it is peripherally associated with early endosomes. These results, together with our finding that Hrs is homologous to Vps27p, a protein essential for protein traffic through a prevacuolar compartment in yeast, suggest that Hrs is involved in vesicular transport through early endosomes.

Many eukaryotic cell surface proteins are bound to the membrane via a glycosylphosphatidylinositol (GPI) anchor. Assembly of the GPI anchor precursor is a sequential addition of components to phosphatidylinositol (PI) in the endoplasmic reticulum (ER). The first step is the transfer of N-acetylglucosamine (GlcNAc) to PI from UDP-GlcNAc to generate GlcNAc-PI. This simple step, however, is regulated by at least three genes because in both mammals and yeasts, there are three mutants of different complementation classes. To clarify this complexity, we analyzed the products of two cloned human genes, PIG-A and PIG-H. Here we demonstrate 1) that PIG-A is an ER transmembrane protein with a large cytoplasmic domain that has homology to a bacterial GlcNAc transferase and a small lumenal domain; 2) that PIG-H is a cytoplasmically oriented, ER-associated protein; and 3) that they form a protein complex. We also show that part of the small lumenal domain of PIG-A plays an essential functional role in targeting itself to the rough ER. Taken together with the cytoplasmic orientation of GlcNAc-PI, these results indicated that PIG-A and PIG-H are subunits of the GPI GlcNAc transferase that transfers GlcNAc to PI on the cytoplasmic side of the ER.

A mitochondrial half-type ATP-binding cassette (ABC) protein, ABC7, plays a role in iron homeostasis in mitochondria, and defects in human ABC7 were shown to be responsible for the inherited disease X-linked sideroblastic anemia/ataxia. We examined the role of ABC7 in the biosynthesis of heme in erythroid cells where hemoglobin is a major product of iron-containing compounds. RNA blots showed that the amount of