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    • br Structure of V ATPase V

    2022-11-15


    Structure of V-ATPase V-ATPase is a highly conserved multi-subunit enzyme that uses energy from ATP hydrolysis to transport protons across membranes [2], [3], [4]. It consists of two major functional domains, V1 and V0 (Fig. 1). The former has eight different subunits (A, B, C, D, E, F, G, and H) and contains three catalytic sites for ATP hydrolysis formed by the A and B subunits (A3B3). The membrane-bound V0 domain is responsible for proton translocation across membranes; in yeast, it contains up to six subunits: a, d, and e, and the proteolipids (c, c′, and c″) [5]. Proteolipids are highly conserved, small four-pass transmembrane proteins that have both termini in the organelle lumen and form a ring-like structure. Six proteolipid subunits have been identified in the ring based on chemical and cryoelectron microscopy analyses of the bovine-coated vesicle complex and an 2797 microscopy analysis of the Nephrops norvegicus c ring [2]. Several accessory proteins are associated with the V-ATPase and are essential for the assembly of the pump complex [6], [7]. One of these, Ac 8–9 (Fig. 1), is a truncated form of the (pro)renin receptor encoded by ATP6ap2. The truncated carboxy terminal fragment M8.9 was found to be associated with V-ATPase [8], and a recent genetic study of mouse Atp6ap2 suggests that the full-length protein is essential for complex assembly [6], [9], [10]. M8.9 is likely generated by furin cleavage and is non-functional [11]. As discussed below, the Atp6ap2 protein provides lines of hot issues, as its function is implicated in multiple signaling cascades.
    V-ATPase in signaling and endocytosis As the primary proton pump that acidifies endosomes and lysosomes, V-ATPase has been intensively studied for its role in endocytosis [12], [13]. Endosomes and lysosomes constitute a highly dynamic intracellular network that enables the internalization of extracellular molecules along with a portion of the cell membrane for degradation, modification, and recycling. This process of endocytosis provides a mechanism for renewing and modifying cell surface and extracellular components and is common to all eukaryotes. Inhibiting endosomal acidification with V-ATPase-specific inhibitors or acidotropic agents affects endocytosis, vesicle trafficking, and the processing of secretory and lysosomal proteins [14]. This is supported by genetic evidence; for instance, depletion of V-ATPase in Atp6v0c mutant mice (lacking the only structural gene for the proteolipid c subunit) affects luminal acidification and endocytosis. In addition, V-ATPase-deficient cells exhibit abnormal Golgi morphology, especially in the trans-region. Genetic deletion of Atp6v0c results in severe developmental defects in early embryogenesis at embryonic day 5 [15]. V-ATPase is also involved in a variety of acidification-independent processes [3], [4]. The best example is vacuolar assembly, in which membrane-encircled lysosome-associated organelles fuse to form larger compartments. This involves a series of biological and physical events that include recognition of distinct membrane organelles followed by membrane fusion, which ultimately leads to the formation of a single compartment enclosed by a continuous membrane. At the final step of fusion, the trans complex formed by the V0 sectors on the paired membranes provides the initial point of contact between the two membranes [16], [17], [18]. This step is thought to be independent of canonical V-ATPase function, i.e., proton translocation and acidification. Other membrane fusion events such as the secretion of insulin-containing granules and neurotransmitter release at the synapse also require V-ATPase, but these fusion events were observed when luminal acidification was inhibited with bafilomycin, a V-ATPase-specific inhibitor, or using proton translocation defective mutant [19], [20]. On the other hand, a controversial observation is also reported: the physical presence of the V-ATPase is not sufficient, but H+-translocation and resulting vacuole acidification are required for vacuole fusion in yeast cells [21]. To elucidate these questions, it may be necessary to use mutations in V-ATPase V0 subunits that eliminate proton translocation activity without affecting assembly or targeting of the resulting V-ATPase [21]. Nevertheless, the essential role of V-ATPase in membrane fusion is undoubtedly.