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Eukaryotic vacuolar-type H؉ -ATPases (V-ATPases) are regulated by the reversible disassembly of the active V 1 V 0 holoenzyme into a cytosolic V 1 complex and a membrane-bound V 0 complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V 1 subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V 1 complex but not as a part of the V 1 V 0 holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V 1 complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3,5-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V 1 V 0 holoenzyme. Vacuolar type Hϩ -ATPases (V-ATPases) 3 are the most versatile proton pumps, being common to all eukaryotic organisms, and are found in endomembrane systems and in the plasma membrane (1-3). V-ATPases are multi-subunit transporters composed of a catalytic ATP-hydrolyzing V 1 complex (Ϸ550 kDa), which resides on the cytoplasmic side of the membrane, and a membrane-bound proton-translocating V 0 complex (Ϸ250 kDa). V-ATPase-dependent proton pumping is essential for cellular pH homeostasis and creates an electrochemical proton gradient that energizes secondary transport mechanisms in a wide variety of organelles and membrane systems. Acidification of organelles by V-ATPase activity is crucial to various cellular processes such as neurotransmitter uptake into synaptic vesicles, intracellular protein trafficking, and the secretion and activation of lysosomal enzymes for protein processing and degradation (4 -7). Located in the plasma membrane of specialized cells, V-ATPases are involved in processes such as cation secretion, bone resorption, renal acidification, and osmoregulation (8 -16). With respect to this diversity of function, mutations in genes encoding V-ATPase subunits obviously lead to several diseases, e.g. osteopetrosis (17) or renal tubular acidosis (18).Several mechanisms have been proposed for the regulation of V-ATPase activity (3). The most prominent and physiologically relevant mechanism is the reversible disassembly of the V-ATPase holoenzyme i...

Reversible assembly of the V0V1 holoenzyme from V0 and V1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H ؉ -ATPases (V-ATPases) in animal cells. In the blowfly (Calliphora vicina) salivary gland, V-ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the cAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that cAMP, Sp-adenosine-3 ,5 -cyclic monophosphorothioate, or forskolin, similar to serotonin, cause VATPase-dependent luminal acidification. In addition, V-ATPasedependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V1 components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca 2؉ , our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus.regulation ͉ translocation ͉ secretion T he vacuolar-type H ϩ -ATPases (V-ATPases) are multisubunit heteromeric complexes that are organized into two domains, designated V 0 and V 1 (1-4). V 0 forms a membranespanning proton-translocating complex; in yeast, it is composed of at least five different subunits termed a, c, cЈ, cЉ, and d, of which subunit c binds bafilomycin A 1 , a specific inhibitor of V-ATPases (5-8). The V 1 sector is attached to the cytoplasmic side of the V 0 sector, consists of at least eight different subunits termed A-H, and contains catalytic and noncatalytic ATPbinding sites. V-ATPase is vital for almost every eukaryotic cell and fulfils a variety of functions. On intracellular acidic membrane systems, such as endosomes, lysosomes, and synaptic vesicles, these proton pumps are involved in protein sorting during biosynthetic and endocytotic pathways, zymogen activation, and transmitter uptake, respectively (3, 4). V-ATPase molecules in the plasma membrane of animal cells, especially on the apical plasma membrane of epithelial cells, contribute to intracellular pH homeostasis, extracellular acidification, or alkalinization, or they energize the plasma membrane for secondary transport processes (3, 4).In some cells, V-ATPase requires a considerable amount of energy. For reasons of economy, it is thus favorable if V-ATPase activity is adapted to the physiological needs of the cell. Several regulatory mechanisms have been identified (1,2,4,9). One of these is the reversible dissociation of the V 1 sector from the V 0 sector, as revealed by experiments performed in yeast, midgut epithelial cells of the tobacco hornworm Manduca sexta, mammalian dendritic cells, and renal epithelial cells (10-16). In th...

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SUMMARYSecretory activity in blowfly salivary glands is activated by the hormone serotonin. We have investigated the distribution and activity of two cation pumps that are possibly involved with transepithelial ion transport, i.e. Na+/K+-ATPase and vacuolar-type H+-ATPase(V-ATPase). By immunofluorescence labelling of secretory cells,Na+/K+-ATPase was localized on the basolateral plasma membrane and V-ATPase on the highly folded apical membrane. Activities of both ATPases were probed in salivary gland homogenates by applying specific inhibitors for these ion pumps, namely ouabain and bafilomycin A1. In control glands, bafilomycin-A1-sensitive V-ATPase activity and ouabain-sensitive Na+/K+-ATPase activity accounted for 36% and 19%, respectively, of the total ATPase activity. V-ATPase activity increased approximately twofold after stimulation with serotonin, whereas Na+/K+-ATPase activity was not significantly affected. Biochemical assays provided evidence that the serotonin-induced activation of V-ATPase activity was accompanied by a recruitment of peripheral V1subunits from the cytosol to the plasma membrane, indicative of the assembly of V0V1 holoenzymes.These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical `potassium pump' that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K+/nH+ antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V0V1holoenzymes might constitute a regulatory mechanism for the control of pump activity.

Microvillar photoreceptors of invertebrates exhibit a light-induced rise in the intracellular concentration of free calcium (Cai) that results in part from release of calcium from an intracellular compartment. This light-induced release of calcium appears to result from a cascade of reactions that involve rhodopsin, a GTP-binding protein and a phospholipase-C which releases inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) from the plasma membrane; the Ins(1,4,5)P3 acts to release calcium from smooth endoplasmic reticulum. In the ventral photoreceptor of the horseshoe crab Limulus polyphemus not all of the endoplasmic reticulum is subject to calcium release by Ins(1,4,5)P3. Only endoplasmic reticulum in the light-sensitive region of the cell is competent to release calcium in response to Ins(1,4,5)P3. The release of calcium by Ins(1,4,5)P3 in ventral photoreceptors appears to be subject to feedback inhibition through elevated Cai. We suggest that this feedback inhibition contributes to sensory adaptation in the photoreceptor and may account for oscillatory membrane responses sometimes observed with large injections of Ins(1,4,5)P3.

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We have reinvestigated the morphology of the salivary glands in the cockroach, Periplaneta americana, by light, electron, and confocal laser scanning microscopy. All secretory acini have a uniform structural layout. They consist of three cell types: peripheral cells, central cells, and duct cells. One pair of peripheral cells forms the base of each acinus. The central cells, arranged concentrically in a fourfold symmetry around the most proximal part of the acinar ducts, lie next downstream. In every acinus, duct cells accompany the central cells and form a thin sheet on the apical surface of the latter. This apical lining of the duct cells is regularly fenestrated, and the central cells secrete the contents of their secretory vesicles only through these openings into the lumen of the ducts. Peripheral cells and central cells are never in direct physical contact, because, apically, extensions of the inner acinar duct cells intervene between the cells. Basally, thin extensions of the basement membrane separate the cell types. We have found no morphological evidence for the existence of electrical coupling (gap junctions) between the saliva-producing cells. Our ultrastructural data support the view that the peripheral cells are responsible for water and electrolyte transport, whereas only central cells secrete the proteinaceous components of the saliva. We have found that the duct cells distal to the acini are also specialized for ion and water transport. They have a prominent basal labyrinth containing numerous mitochondria and a highly folded apical surface. The folded apical membrane surface is coated with electron-dense particles on its cytoplasmic side; these particles are probably portasomes. Our investigation provides morphological evidence that the duct cells distal to the secreting acini are able to modify primary saliva.