Vacuolar ATPase in Phagosome-Lysosome Fusion

Sandra Kissing,Christina Hermsen,Urska Repnik,Cecilie Kåsi Nesset,Kristine Von Bargen,Gareth Griffiths,Atsuhiro Ichihara,Beth S Lee,Michael Schwake,Jef De Brabander,Albert Haas,Paul Saftig

doi:10.1074/jbc.m114.628891

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.

Highlights

The vacuolar Hϩ-ATPase complex is thought to contribute to membrane fusion
Beads that have not been taken up were stained with anti-IgG antibodies coupled to Alexa Fluor 594 for 1 min, and the samples were processed for immunofluorescence staining of the marker proteins early endosome antigen 1 (EEA1) or LAMP-2, respectively
V0 Subunit a3 Deficiency Despite Normal pH of the Lysosome— In a genetic-based approach to analyze the role of the v-ATPase subunit a3 in membrane fusion, we used cells from Tcirg1oc/oc mice, which are homozygous for the osteosclerosis mutation and naturally lack the transmembrane a3-subunit of the v-ATPase [24]

Summary

Background

The vacuolar Hϩ-ATPase complex is thought to contribute to membrane fusion. Results: v-ATPase complex knock-out experiments in mice revealed that its absence does not affect phagosome-lysosome fusion. In the past few years evidence has accumulated that the transmembrane V0 part of the v-ATPase, without participation of the V1 sector, can play active and critical roles in membrane fusion along the endocytic and exocytic pathways independent of its proton-translocating activities Such evidence stems from experiments on synaptic vesicle exocytosis in Drosophila [13], secretion in Caenorhabditis [14], osteoclast fusion [15], and vesicle fusion in zebrafish microglia cells after ingestion of neuron-derived apoptotic bodies [16]. As tools we used the Tcirg1oc/oc mouse line and conditionally deleted Atp6ap knock-out mice Cells derived from these mice either lacked the v-ATPase subunit a3 or showed an almost complete absence of the v-ATPase V0 sector yet allowed phagosome-lysosome as well as endosome-lysosome fusion to progress. All animal experiments were conducted in agreement with local guidelines for the use of animals and their care

Experimental Procedures

Results

Discussion