Vacuolar ATPase in Phagosome-Lysosome Fusion [Membrane Biology]

April 22nd, 2015 by Kissing, S., Hermsen, C., Repnik, U., Nesset, C. K., von Bargen, K., Griffiths, G., Ichihara, A., Lee, B. S., Schwake, M., De Brabander, J., Haas, A., Saftig, P.

The vacuolar H+-ATPase (v-ATPase) complex is instrumental to establish and maintain 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 proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts (MEF) 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 MEF 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.