Vacuolar proton-translocating ATPase pumps consist of two domains, V 1 and V o . Subunit d is a component of V o located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59 -72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V 1 V o , mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N-and C-terminal deletions of various lengths were inhibitory and gradually desta- Viral infections, cancer, osteoporosis, and renal tubular acidosis are some of the human disease states associated with the V-ATPase 2 function. V-ATPases are ATP-driven proton pumps present in Golgi, endosomes, lysosomes, and vacuoles, where they are responsible for maintaining the acidic luminal pH essential for receptor-mediated endocytosis, zymogen activation, and protein sorting (1-4). In addition to the endogenously distributed V-ATPases, some cells contain V-ATPases at the plasma membrane, where they pump protons from the cytosol to the extracellular milieu. V-ATPase proton transport across the plasma membrane is essential for bone resorption, urinary acidification, sperm maturation, and neurotransmitter sequestration (3).V-ATPases are related to F-ATP synthases (5), and both protein complexes work as molecular motors (6 -9). V-ATPases, however, work exclusively in the direction of ATP hydrolysis in vivo. V-ATPases consist of two domains, V 1 and V o , similar to the F 1 and F o domains found in ATP synthases (1-4). Eight different subunits (A-H) compose V 1 , which is peripherally attached to the membrane and hydrolyzes ATP (1-4). Six different subunits (a, c, cЈ, cЉ, d, and e) associate to form V o , which holds V 1 at the membrane and forms the path to transport protons (1-4). V 1 and V o subunits contribute to the formation of one central stalk and two or three peripheral stalk structures that connect a proteolipid ring (made of subunits c, cЈ, and cЉ) in V o and the catalytic core of V 1 (a hexamer of three subunits A alternating with three subunits B) (10, 11).The organization of the central and peripheral stalks is essential for structural and functional coupling of ATP hydrolysis and proton transport. It is proposed that during catalysis, ATP hydrolysis at the V 1 hexamer A 3 B 3 drives rotation of a rotor (central stalk made of subunits D, F, and d connected to the proteolipid ring) (6, 12). Six essential glutamates are protonated in the ring when protons are transferred from the cytosol via two half-tunnel structures formed in the stationary subunit a at the membrane (13-15). By connecting the stationary subunits (A 3 B 3 and a), the peripheral stalk(s) (subunits C, E, G, H, and the N terminus of subunit a) wo...

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membranebound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V 0 subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V 1 and V 0 (؊d) that failed to form V 1 V 0 , and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V 1 V 0 assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V 1 V 0 was delivered to the vacuole and active. It retained 63-71% of the wild-type activity and was responsive to glucose. Tether-less V 1 V 0 disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V 1 subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V 0 assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6. V-ATPase4 proton pumps are highly conserved proteins fundamental for pH homeostasis (for review, See Refs. 1-6). Located in the endomembrane system, V-ATPases establish and maintain the low pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting (for review, see Refs. 1-3). Cells specialized for active proton secretion, like kidney epithelial cells and osteoclasts, also express V-ATPases at the plasma membrane, where they transfer protons from the cytosol to the extracellular milieu (4, 5). In the kidney, plasma membrane V-ATPases of the intercalated cells are critical for regulation of the systemic acid-base balance (5, 6). Mutations in human kidney V-ATPase cause distal-renal tubular acidosis (6). V-ATPases at the plasma membrane of osteoclasts are essential for bone resorption, and mutations result in osteopetrosis, a disease characterized by thickening of the bones (1, 4, 7). Complete loss of V-ATPase activity is lethal in eukaryotes other than fungi (3).V-ATPases are multisubunit complexes that consist of two domains, V 1 (peripheral) and V 0 (membrane-bound) (1, 2). Each of the subunits in the V-ATPase complex is critical for function and V 1 V 0 assembly (8). Deletion of a peripheral V 1 subunit leads to disruption of the entire V 1 domain in yeast. Loss of a V 0 subunit does not affect V 1 assembly but disrupts the entire V 0 domain, which also prevents V 1 from associating with the membrane. An exception is subunit a for which two functional isoforms (Vph1, Stv1) exist in yeast (9). Disruption of subunit a requi...

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The formation of 3-arylbenzaldehydes through rearrangement of 3-aryl-8-oxabicyclo[3.2.1]oct-2-en-7-ones is reported, together with various rearrangement reactions of 2-aryl-8-oxabicyclo[3.2.1 loct-6-en-3-ones, and a novel approach to the natural product acamelin.

On page 1544, the structure of compound (6) should be as shown below.Electronically typeset and printed by Heffers Printers Ltd,

The corresponding oxyallyl cations formed from the bromoketones (I) the synthesis of which is described react with furan (II) in the presence of trifluoroethanol (III) to give the oxabicyclooctenones (IV) or (VI) as well as the side‐products (V) (= products from a Favorskii rearrangement) or (VII).

Die aus Furan (II) in der skizzierten Weise zugänglichen Aryl‐oxabicyclooctenone (VII) lagern sich in die Arylbenzaldehyde (VIII) um.