Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70-and 60-kDa subunits of the vacuolar H+-ATPase are -25% identical to the .8 and a subunits, respectively, of the eubacterial-type FOFj-ATPases. We now report that the same vacuolar H+-ATPase subunits are -50% identical to the a and 13 subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the FOFj-ATPase P subunit but is present in the a subunit of Sulfolobus. Since the two types of subunits (a and 13 subunits; 60-and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.Recently, attention has focused on the evolutionary relationships among the H+-ATPases, particularly the F0F1-ATPases (F-type) and vacuolar (V-type) H+-ATPases. F-and VATPases exhibit a number of structural and functional similarities (1-4). Both are large, multisubunit enzymes (=500 kDa) composed of a water-soluble catalytic sector and an integral membrane proton channel complex. Each hydrophilic sector contains three copies of the catalytic subunit (F-ATPase (3 subunit or V-ATPase 70-kDa subunit), three copies of a regulatory subunit (F-ATPase a subunit or V-ATPase 60-kDa subunit), and one copy each of several minor subunits (4). Sequences obtained for several eukaryotic V-ATPase 70-and 60-kDa subunits confirmed that the Fand V-type H+-ATPases are indeed homologous (5-9). However, the low overall similarity (25%) and the presence of a large stretch of nonhomologous sequence in the 70-kDa subunit (5) suggest that they diverged early in evolution. Consistent with this view, sequences obtained for the two major subunits of the membrane H+-ATPase of Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium), indicated that the "archaebacterial-type" H+-ATPase is only distantly related to the eubacterial-type F-ATPases (10, 11). In this joint communication from four of the laboratories involved, we show that the H+-ATPase of S. acidocaldarius belongs in the V-ATPase class of proton pumps. The implications for the origin of eukaryotes are discussed. MATERIALS AND METHODSTo determine the evolutionary relationships among the different H+-ATPases, protein or DNA sequences coding for the two major subunits or parts of these subunits were aligned, ...

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Abstract. The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37°C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including a-mannosidase I1, [3-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intraGolgi pH (priG), which was calibrated in situ with ionophores. In intact Vero cells, prig averaged 6.45 +__ 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases. pH6 remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H ÷ antiport. Similarly, priG was unaffected by acute changes in cytosolic calcium. Furthermore, prig recovered quickly toward the basal level after departures imposed with weak bases. These fndings suggest that pHG is actively regulated, despite the presence of a sizable H ÷ "leak" pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.

Vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular compartments in eukaryotic cells. V-ATPases possess a subunit of approximate molecular mass 100 kDa of unknown function that is composed of an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain. To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes. Ten charged and twelve polar residues located in the seven putative transmembrane helices in the COOH-terminal domain of the molecule were individually changed, and the effects on proton transport, ATPase activity, and assembly of the yeast V-ATPase were measured. Two mutations (R735L and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly reduced levels of the 100-kDa subunit in the vacuolar membrane, suggesting that these mutations affected stability of the 100-kDa subunit. Two mutations, D425N and K538A, in transmembrane helix 1 and at the border of transmembrane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complexes that did assemble. Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, have significantly greater effects on activity than on assembly, with proton transport and ATPase activity inhibited 40-60%. One mutation, E789Q, in transmembrane helix 7, virtually completely abolished proton transport and ATPase activity while having no effect on assembly. These results suggest that the 100-kDa subunit may be required for activity as well as assembly of the V-ATPase complex and that several charged residues in the last four putative transmembrane helices of this subunit may play a role in proton transport.

Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous ␣ 2 ␤ 1 integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated ␣ 2 ␤ 1 integrin after initial bead binding. ␤-Actin and gelsolin associated transiently with beads (0 -30 min) followed by LAMP-2 (60 -240 min) and cathepsin B (30 -240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30 -120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.Phagocytosis is central to the uptake and degradation of microorganisms as well as damaged or senescent cells and is therefore an essential process in host defense, tissue remodeling, and inflammation. Although "professional" phagocytic cells such as macrophages and neutrophils have been studied in considerable depth (1), several types of nonphagocytic cells such as epithelial cells and fibroblasts can internalize particles and matrix proteins in vivo and in culture. Collagen fibril phagocytosis is thought to be an important pathway for physiological degradation of extracellular matrix in mature connective tissues (2). Uterine, wounded dermal, and periodontal connective tissues exhibit rapid physiological turnover of matrix proteins, processes that are mediated by the intracellular degradation pathway (3). While in inflamed periodontal sites, extracellular matrix metalloproteinases are believed to be responsible for the bulk degradation of connective tissues (4), in normal turnover, fibroblasts are thought to use solely the intracellular vacuolar system for focal proteolysis of collagen (3,5). However, at present, the vacuolar system that mediates intracellular collagen degradation is incompletely characterized.In professional phagocytes, the phagocytic process is initiated by binding of particles to receptors on the plasma membrane, an event that subsequently generates a phagocytic signal (6). In fibroblasts, the initial internalization of intracellular collagen degradation is a specific process that is mediated by the adhesive interactions between ligand and collagen receptors (i.e. ␣ 2 ␤ 1 integrins; Ref. 7). However, little is known about the regulation of the downstream events and...