title: Vacuolar ATPase in phagosome-lysosome fusion.
abstract: The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.
authors.affiliation: From the Institute of Biochemistry, Christian-Albrechts-University of Kiel, D-24098 Kiel, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Kissing
authors.affiliation: Institute for Cell Biology, Friedrich-Wilhelms University, D-53121 Bonn, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Hermsen
authors.affiliation: Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Urska
authors.initials: U
authors.lastname: Repnik
authors.affiliation: Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Cecilie Kåsi
authors.initials: CK
authors.lastname: Nesset
authors.affiliation: Institute for Cell Biology, Friedrich-Wilhelms University, D-53121 Bonn, Germany.
authors.forename: Kristine
authors.initials: K
authors.lastname: von Bargen
authors.affiliation: Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Gareth
authors.initials: G
authors.lastname: Griffiths
authors.affiliation: Department of Medicine II, Tokyo Women's Medical University, Tokyo 162-866, Japan.
authors.forename: Atsuhiro
authors.initials: A
authors.lastname: Ichihara
authors.affiliation: Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, Ohio 42210.
authors.forename: Beth S
authors.initials: BS
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Biochemistry III, University of Bielefeld, D-33615 Bielefeld, Germany, and.
authors.forename: Michael
authors.initials: M
authors.lastname: Schwake
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
authors.forename: Jef
authors.initials: J
authors.lastname: De Brabander
authors.affiliation: Institute for Cell Biology, Friedrich-Wilhelms University, D-53121 Bonn, Germany, albert.haas@uni-bonn.de.
authors.forename: Albert
authors.initials: A
authors.lastname: Haas
authors.affiliation: From the Institute of Biochemistry, Christian-Albrechts-University of Kiel, D-24098 Kiel, Germany, psaftig@biochem.uni-kiel.de.
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D008297:Male; D008561:Membrane Fusion; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D000093342:Prorenin Receptor
chemical_list: D006180:Proton-Translocating ATPases; D011956:Receptors, Cell Surface; D025262:Vacuolar Proton-Translocating ATPases; C583530:ATP6AP2 protein, mouse; D000093342:Prorenin Receptor
keywords: ATP6AP2; lysosomal acidification; lysosome; membrane fusion; phagocytosis; phagosome maturation; vacuolar ATPase