VacuolarATPaseinPhagosome-LysosomeFusiontein (DPGYDSIIYRMTNQ, Pineda-abservice). ExperimentalAnimals Mice with loxP sites flanking exon 2 of the Atp6ap2 gene have been described previously

Vacuolar ATPase in Phagosome-Lysosome FusionReceived for publication, November 29, 2014, and in revised form, April 13, 2015 Published, JBC Papers in Press, April. 22, 2015, DOI 10.1074/jbc.M114.628891

Sandra Kissing‡1, Christina Hermsen§1, Urska Repnik¶, Cecilie Kåsi Nesset¶, Kristine von Bargen§, Gareth Griffiths¶,Atsuhiro Ichihara�, Beth S. Lee**, Michael Schwake‡‡, Jef De Brabander§§, Albert Haas§2, and Paul Saftig‡3

From the ‡Institute of Biochemistry, Christian-Albrechts-University of Kiel, D-24098 Kiel, Germany, §Institute for Cell Biology,Friedrich-Wilhelms University, D-53121 Bonn, Germany, ¶Department of Biosciences, University of Oslo, 0316 Oslo, Norway,�Department of Medicine II, Tokyo Women’s Medical University, Tokyo 162-866, Japan, **Department of Physiology and CellBiology, The Ohio State University College of Medicine, Columbus, Ohio 42210, ‡‡Department of Chemistry, Biochemistry III,University of Bielefeld, D-33615 Bielefeld, Germany, and §§Department of Biochemistry, University of Texas Southwestern MedicalCenter, Dallas, Texas 75390

Background: The vacuolar H�-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-lysosomefusion.Conclusion: Participation of v-ATPase in phagosome-lysosome fusion is unlikely.Significance: Fusion between lysosomes/late endosomes and phagosomes is not controlled by the v-ATPase.

The vacuolar H�-ATPase (v-ATPase) complex is instrumen-tal in establishing and maintaining acidification of some cellularcompartments, thereby ensuring their functionality. Recently ithas been proposed that the transmembrane V0 sector ofv-ATPase and its a-subunits promote membrane fusion in theendocytic and exocytic pathways independent of their acidifica-tion functions. Here, we tested if such a proton-pumping inde-pendent role of v-ATPase also applies to phagosome-lysosomefusion. Surprisingly, endo(lyso)somes in mouse embryonicfibroblasts lacking the V0 a3 subunit of the v-ATPase acidifiednormally, and endosome and lysosome marker proteins wererecruited to phagosomes with similar kinetics in the presence orabsence of the a3 subunit. Further experiments used macro-phages with a knockdown of v-ATPase accessory protein 2(ATP6AP2) expression, resulting in a strongly reduced level ofthe V0 sector of the v-ATPase. However, acidification appearedundisturbed, and fusion between latex bead-containing phago-somes and lysosomes, as analyzed by electron microscopy, waseven slightly enhanced, as was killing of non-pathogenic bacte-ria by V0 mutant macrophages. Pharmacologically neutralizedlysosome pH did not affect maturation of phagosomes in mouseembryonic cells or macrophages. Finally, locking the two largeparts of the v-ATPase complex together by the drug saliphenyl-halamide A did not inhibit in vitro and in cellulo fusion of pha-gosomes with lysosomes. Hence, our data do not suggest afusion-promoting role of the v-ATPase in the formation ofphagolysosomes.

A phagocytic compartment (phagosome) is formed when aparticle is ingested through receptor-ligand interaction into a

plasma membrane invagination. Newly formed phagosomesare not static compartments but rather acquire degradative andmicrobicidal properties through a complex series of interac-tions with endomembranes. This process, collectively termedphagosome maturation, culminates in the fusion of phago-somes with lysosomes, yielding a strongly acidic (pH 4.5–5.0)hybrid organelle enriched in hydrolytic enzymes and antimi-crobial peptides that promotes killing and degradation of inter-nalized microorganisms (1, 2). The main role of phagocytosis isthe delivery of microbial invaders and apoptotic bodies tophagolysosomes. For most microbes the acidic, hydrolyticallycompetent environment of the eventually formed phagolyso-some is sufficient to kill them. The strong acidification of(phago-)lysosomes is a hallmark feature of the endocytic andphagocytic pathways and is generated by a large multiproteincomplex, the vacuolar ATPase (v-ATPase)4 (3, 4). Acidificationis required for trafficking of endosomes (5) and for killing ofingested microorganisms (6). The precise subcellular origin ofall vesicles, which can deliver v-ATPases to phagosomes is,however, not clear. The v-ATPase complex is formed by at least14 subunits that are organized in two large subparticles; ofthese, the V0 sector contains transmembrane proteins thatform the proton channel, whereas the V1 sector is cytosolic andis responsible for the ATPase activity (7). How exactly the sub-units are assembled within the endoplasmic reticulum anddelivered to lysosomes is not understood. However, it has beenshown that “accessory subunits,” such as the v-ATPase acces-sory protein 2 (ATP6AP2 (8)), are important and that a failureto properly assemble the V0 portion leads to its degradation.Hence, it is not surprising that mutated v-ATPase subunits

* This work was supported by Deutsche Forschungsgemeinschaft GrantsGRK1459 (to P. S. and S. K.) and SPP1580 (to A. H., G. G., and P. S).

1 Both authors equally contributed to this work.2 To whom correspondence may be addressed. E-mail: albert.haas@

uni-bonn.de.3 To whom correspondence may be addressed. E-mail: [email protected]

kiel.de.

4 The abbreviations used are: v-ATPase, vacuolar ATPase; ATP6AP2, ATPase,H�-transporting lysosomal accessory protein 2; cKO, conditional knock-out; EEA1, early endosome antigen 1; LAMP-1, lysosomal-associated mem-brane protein 1; LAMP-2, lysosomal-associated membrane protein 2; LBPs,latex bead-containing phagosomes; SaliPhe, saliphenylhalamide A; Fc�RII,Fc-� receptor II; BMDM, bone marrow-derived macrophage; MEF, mouseembryonic fibroblast.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 22, pp. 14166 –14180, May 29, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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cause many severe illnesses such as the progression of cancerand bone disorder (9, 10).

In the past few years evidence has accumulated that thetransmembrane V0 part of the v-ATPase, without participationof the V1 sector, can play active and critical roles in membranefusion along the endocytic and exocytic pathways independentof its proton-translocating activities. Such evidence stems fromexperiments on synaptic vesicle exocytosis in Drosophila (13),secretion in Caenorhabditis (14), osteoclast fusion (15), andvesicle fusion in zebrafish microglia cells after ingestion of neu-ron-derived apoptotic bodies (16). The original observation ofan acidification-independent role in membrane fusion arosefrom studies on yeast homotypic vacuole fusion (11, 12). How-ever, a recent study using the same model organelle proposed amodel in which the major role of the v-ATPase complex wouldbe for vacuolar acidification (17).

To address the question in how far fusion events depend onthe presence of the v-ATPase in mammalian cells, we made useof phagosome-lysosome fusion, as lysosomes are highly acidic,contain large quantities of v-ATPase, and fuse with endocyticvesicles. As tools we used the Tcirg1oc/oc mouse line and condi-tionally deleted Atp6ap2 knock-out mice. Cells derived fromthese mice either specifically lacked the v-ATPase subunit a3 orshowed an almost complete absence of the v-ATPase V0 sectoryet allowed phagosome-lysosome as well as endosome-lyso-some fusion to progress. Also, locking the V0 and V1 sectors ofv-ATPase together did not affect phagosome maturation andneither did pharmacologic alkalization of lysosome pH.

Experimental Procedures

Antibodies

The following antibodies were used for protein detection inWestern blot and immunofluorescence analyses: anti-ATP6AP2 N-terminal (HPA003156, Sigma), anti-�-actin(A2066, Sigma), anti-EEA1 (#C45B10, Cell Signaling Technol-ogy or #AV30074, Sigma), anti-cathepsin D (clone sII-10, a kindgift from Dr. S. Höning, University Cologne, Germany), anti-cathepsin L (#AF1515, R&D Systems), anti-glyceraldehyde-3-phosphate-dehygrogenase (GAPDH, clone FL335, #sc-25778,Santa Cruz Biotechnology), anti-LAMP1 (1D4B, DSHB), anti-LAMP-2 (Abl93, DSHB), anti-Myc (clone 9B11, #2276, CellSignaling Technology), anti-transferrin receptor (TIB-219,ATCC), anti-V0 a1 (18), anti-V0 a2 (#ab96803, Abcam), anti-V0a3 (generous gift from Dr. T. Jentsch, FMP, Berlin, Germany),anti-V0 d1 (#18274 –1-AP, Proteintech group), anti-V1 A(kindly supplied by Dr. Shoji Ohkuma, Kanazawa University,Japan (19), anti-V1 E1 (20), rabbit polyclonal antibody to bovineB-subunit of vacuolar ATPase (Yoshinori Moriyama, OkayamaUniversity, Japan), and anti-V1 B2 (clone D2F9R, #14617, CellSignaling Technology). Secondary antibodies conjugated tohorseradish peroxidase, Alexa Fluor 488, Alexa Fluor 594, orAlexa Fluor 647 were purchased from Dianova and Life Tech-nologies. An anti-V0 c antibody was raised in rabbit against asynthetic peptide corresponding to residues 26 – 44 of themurine protein (CSAMGAAYGTAKSGTGIAAM) and puri-fied by affinity chromatography against the immobilized pep-tide (Pineda-abservice). To generate an anti-ATP6AP2 C-ter-

minal antibody, rabbits were immunized with a syntheticpeptide corresponding to residues 332–345 of the murine pro-tein (DPGYDSIIYRMTNQ, Pineda-abservice).

Experimental Animals

Mice with loxP sites flanking exon 2 of the Atp6ap2 genehave been described previously (21). Atp6ap2Flox/Flox femalemice were bred with male mice, expressing the Cre recombi-nase under the control of an inducible Mx1 promotor (22) orthe LysM promotor (23) to yield Atp6ap2Flox/Y Cretg/� mice.Further breeding with homozygous Atp6ap2Flox/Flox femalemice resulted in Atp6ap2Flox/Flox/Cretg/� or Atp6ap2Flox/Y/Cretg/� animals (conditional knock-out (cKO)). Littermatesnegative for Mx1-Cre or LysM-Cre, respectively, served as con-trol (wild-type).

Cre expression was induced in 6-week-old Mx1-Cre trans-genic and control mice by intraperitoneal injection of 3 doses of250 �g of polyinosinic-polycytidylic acid (Sigma) within 5 days.Mice were kept for a further 10 days and sacrificed for experi-mental analysis. When required, intraperitoneal injection of 0.5ml of 4% (w/v) Brewer’s thioglycolate solution (Difco/BD Bio-sciences) was performed to enrich peritoneal macrophages, andcells were harvested by peritoneal lavage 3 days later.

We are grateful to Dr. Uwe Kornak, Charité Berlin, Germany,for providing mice carrying the osteosclerotic mutation (oc/oc)in the Tcirg1 locus (24). All animal experiments were con-ducted in agreement with local guidelines for the use of animalsand their care.

Cell Lines and Primary Cell Culture

All cell types were grown in Dulbecco’s modified Eagle’smedium (DMEM) with 4 mM L-glutamine and 4.5 g/liter glu-cose (PAA Laboratories or Sigma) supplemented with 10% (v/v)fetal bovine serum (FBS, PAA Laboratories or Biochrom AG).For maintenance of primary cells, 100 units/ml penicillin and100 �g/ml streptomycin (PAA Laboratories or Sigma) wereadded to the growth media. Cultures were grown at 37 °C in ahumidified 5% CO2 atmosphere condition unless statedotherwise.

Murine embryonic fibroblast were generated from 13–14-day-old embryos of breeding pairs yielding Tcirg1oc/oc (a3-de-ficient) and Tcirg1�/� (wild-type) MEFs. Embryos were decap-itated, and inner organs were removed before single cells wereobtained by trypsin digest. All MEF lines were immortalizedafter three-four passages by transfection with the SV40 Large Tantigen. Where indicated, MEFs were further cotransfectedwith Fc-� receptor II (Fc�RII; kind gift of Dr. Sergio Grinstein)and pcDNA4/TO (Addgene) and selected for stable proteinexpression in the presence of 250 �g/ml zeocin (InvivoGen).MEFs deficient for cathepsin D (CtsD�/�) and cathepsin L(CtsL�/�) have been described before (25, 26).

Murine primary peritoneal macrophages were collected byperitoneal lavage with 8 ml of ice-cold phosphate-bufferedsaline (PBS), centrifuged at 210 � g for 10 min at 4 °C, andresuspended in DMEM with FBS, 100 units/ml penicillin, and100 �g/ml streptomycin for plating. Non-adherent cells wereremoved after 3 h incubation at 37 °C and 5% CO2, and exper-iments were conducted the following day.

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To obtain bone marrow-derived macrophages (BMDMs),long bones (tibia, femur, scapula) from Atp6ap2 cKO and con-trol animals were dissected, and the bone marrow was flushedthrough an 100-�m nylon cell strainer (BD Biosciences) withPBS. BMDMs were cultured in DMEM containing 10 mM

HEPES, 10 mM L-glutamine, 1 mM sodium pyruvate, 100units/ml penicillin, 100 �g/ml streptomycin, 1% GlutaMAX,10% (v/v) FCS, 5% horse serum, and 30% spent L929 culturesupernatant at 9.5% CO2, and cells were used in experimentalsetups after 7–10 days of differentiation. The murine macro-phage-like cell line RAW264.7 (TIB-71) was from ATCC, andthe murine macrophage-like cell line J774E (27) was kindlydonated by Philip Stahl (Washington University, St. Louis,Missouri).

RNA Extraction, Reverse Transcription, and QuantitativeReal-time-PCR

Total RNA was extracted using the NucleoSpin RNA II kit(Macherey-Nagel), and 0.2–1 �g of RNA was spent for reversetranscription using the RevertAid RT kit and random hexamerprimers (Thermo Fisher Scientific). Specific assays for quanti-tative real-time PCR were created with the Universal ProbeLibrary Assay Design Center, and PCR was performed in aLightcycler 480 II (Roche Applied Science). Relative mRNAexpression was calculated by normalizing Cp values to the log-arithmic average Cp of the most stable house keeping genes(Tuba1a, Hprt1, and Sdha). The resulting DCp values werecompared between genotypes for statistical analyses. Primerefficiency (E) was determined for each PCR by co-measurementof a set of serial cDNA dilutions to obtain E(�DCp) plots describ-ing relative mRNA expression levels.

Western Blotting

Total cell lysates were generated by adding PBS containing1% (v/v) Triton-X-100 and 1� cOmplete Protease InhibitorMixture (Roche Applied Science) to cells on ice for 20 minfollowed by sonication for 2 � 10 s using a Branson Sonifer 450(level 7 in a cup horn, Emerson Industrial Automation) andcentrifugation at 16,000 � g for 10 min at 4 °C. Protein concen-trations of the resulting supernatants were measured with thePierce BCA protein assay kit (Thermo Fisher Scientific), andsamples were adjusted to 2 �g of protein/ml. 20 – 40 �g of pro-tein were subjected to SDS-PAGE and analyzed by immuno-blotting. Lysosomes were purified and immunoblotted asdescribed (28) and used at 10 �g of protein per lane.

Subcellular Fractionation

Confluent RAW264.7 macrophage cultures were incubatedfor 2 h in the presence of 10 �M saliphenylhalamide A (SaliPhe)or DMSO and lysed in a Dounce homogenizer with 15 strokesin HB (8.6% (w/v) sucrose, 20 mM HEPES/KOH (pH 7.2), 0.5mM EGTA) containing 1� cOmplete Protease Inhibitor Mix-ture (Roche Applied Science). Total lysates (fraction T) wereseparated into nuclei (fraction P1) and post nuclear superna-tant (PNS, fraction S1) by a 15-min centrifugation step at 960 �g. For further fractionation, PNS were centrifuged at 128,000 �g for 60 min to yield fractions enriched in cytosolic (fraction S2)and membrane-bound (fraction P2) proteins. Pellets and super-

natants were handled in equal volumes. Samples of 10 �l weretaken from each fraction and subjected to Western blotting.

Co-immunoprecipitation

For co-immunoprecipitation analyses, RAW264.7 cells weregrown to almost complete confluence. Where indicated, cellswere preincubated for 2 h with 10 �M SaliPhe or DMSO ascarrier control, and treatment was continued throughout thewhole immunoprecipitation protocol. Cell lysates were gener-ated using lysis buffer (40 mM HEPES (pH 7.4), 12.5 mM EDTA,2.5 mM MgCl2, 10 mM �-glycerophosphate, 10 mM NaF, 0.3%(w/v) CHAPS, 1� cOmplete Protease Inhibitor Mixture (RocheApplied Science)) and centrifuged at 16,000 � g and 4 °C for 10min. Dynabeads Protein G (Life Technologies) were preincu-bated with rotation in 5% (w/v) BSA in lysis buffer for 2 h at 4 °Cto block unspecific binding. 3 mg of soluble protein were pre-cleared of proteins with affinity to the bead matrix material by a2-h incubation with 25 �l of Dynabeads Protein G at 4 °C, beadswere removed, and the lysates were incubated in presence ofprimary antibody overnight at 4 °C with rotation. 50 �l of fresh,blocked Dynabeads were added to the lysate-antibody mix for30 min at room temperature to capture immune complexes.Immunoprecipitated proteins bound to beads were then rinsed3� in lysis buffer containing 150 mM NaCl. 50 �l of SDS-samplebuffer was added per sample, and samples were heated for 5min at 95 °C. 10 �l of each denatured immunoprecipitate wereseparated using 4 –12% NuPAGE Novex Bis-Tris Protein Gels(Life Technologies) and immunoblotted. For detection ofv-ATPase V1 subunit B2, Clean Blot IP Detection Reagent (LifeTechnologies) was used instead of regular anti-rabbit horserad-ish peroxidase to avoid interference of denatured immunopre-cipitation antibody fragments.

Immunofluorescence Analysis

Semi-confluent cultures of cells grown on coverslips werefixed in 4% (w/v) paraformaldehyde solution for 20 min at roomtemperature and permeabilized with 0.2% (w/v) saponin in PBS.Unspecific antibody binding was blocked by preincubating thefixed and permeabilized samples with 10% (v/v) FBS in 0.2%(w/v) saponin in PBS (blocking solution). Antibody staining wasperformed in blocking solution overnight at 4 °C (primary anti-body) and for 1 h at room temperature (secondary antibody) ina humidified chamber. Coverslips were embedded in 17% (w/v)Mowiol 4 – 88 (Calbiochem), 33% (v/v) glycerol, 20 mg/mlDABCO (1,4-diaza-bicyclo-[2,2,2]-octane, Sigma), and 5 �g/mlDAPI (4,6-diamidino-2-phenylindole; Sigma) in PBS. AnOlympus FV1000 confocal laser scanning microscope was usedfor image acquisition.

Visualization of the endocytic pathway was achieved withdextran-Texas Red (Mr 70,000, Life Technologies). Cells grownon coverslips were incubated with 0.5 mg/ml dextran-TexasRed in DMEM containing 1 mg/ml bovine serum albumin(BSA) for 30 min. After rinsing 3 times with PBS and incubationin DMEM � 1 mg/ml BSA for further 3 h, samples were pro-cessed for immunofluorescence analysis as described above. Ina similar approach, the self-quenched fluorochrome DQ-BSARed (Life Technologies) was used to assess proteolytic activi-ties. Therefore, cells were cultured overnight in the presence of



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100 �g/ml DQ-BSA Red in DMEM containing 1 mg/ml BSA,rinsed 3 times with PBS, and processed for fluorescencelabeling.

Analysis of Lysosome pH

Acidic cellular compartments were detected using the acido-tropic dye LysoTracker Red DND-99 (Life Technologies). Cellswere incubated with 333 nM LysoTracker Red in DMEM onlyfor 20 min at 37 °C, fixed in 4% (w/v) paraformaldehyde solu-tion, and embedded or directly analyzed by live cell fluores-cence microscopy.

Ratiometric measurement of lysosome pH was performed asdescribed previously (29, 30). Briefly, endocytic compartmentsof the analyzed cells were loaded with dextran-Oregon Green514 (0.5 mg/ml, 70,000 Mr, Life Technologies) overnight. Toenable pH analysis of late endosomes and lysosomes only, earlycompartments were cleared of label by a 2-h chase in theabsence of the dextran derivate. The addition of 200 nM bafilo-mycin A1 or 10 �g/ml nigericin for 15 min at the end of thischase period was used to collapse the lysosomal pH gradient.Ratiometric imaging was accomplished by exciting samplesalternately at 440 or 488 nm. For an in situ calibration cells weresequentially incubated with K�-rich buffer solutions (145 mM

KCl, 10 mM glucose, 1 mM MgCl2, and 20 mM of either MES oracetate, pH 4.0 – 6.5) including 10 �g/ml K�-ionophore nigeri-cin at the end of each experiment. Lysosome pH was then inter-polated from the generated calibration curve fitted to the Boltz-mann equation.

In Cellulo Latex Bead Phagocytosis Assays

Immunofluorescence Analysis—Latex beads (1.1-�m diame-ter; Sigma) were opsonized with IgG (human or murine; Sigma)overnight at 4 °C to allow uptake by human Fc�RII-expressingMEFs or murine macrophages. Cells were grown to semi-con-fluence on coverslips, and the media were replaced by serum-free DMEM at the point of latex bead administration. To syn-chronize uptake, beads were briefly spun down onto the cells at300 � g for 1 min, and bead uptake was allowed for 15 min at37 °C, 5% CO2. Latex bead excess was washed away with PBS,and fresh DMEM was added. Beads that have not been taken upwere stained with anti-IgG antibodies coupled to Alexa Fluor594 for 1 min, and the samples were processed for immunofluo-rescence staining of the marker proteins early endosome anti-gen 1 (EEA1) or LAMP-2, respectively. In the case of primarymurine macrophages, co-staining with an anti-V0 a3 antibodywas included to assess v-ATPase knockdown efficiency. Whereindicated, 200 nM bafilomycin A1 or 10 �g/ml nigericin wereused to alkalinize lysosomes during the 15-min incubation withlatex beads and during the following chase periods.

A second approach to follow fusion between latex bead-con-taining phagosomes (LBP) and lysosomes in cells was done inRAW264.7 cells. Briefly, endocytic compartments ofRAW264.7 macrophages were loaded with 50 �g/ml BSA-rho-damine overnight. Cells were rinsed with PBS and incubated inthe absence of BSA-rhodamine for 2 h to ensure labeling of lateendosomes and lysosomes only. Treatment with 10 �M SaliPheor DMSO, respectively, was started concurrently with the 2-hchase period and continued until cell lysis. Latex beads (1 �m

diameter, Polysciences) opsonized with murine IgG Fc-frag-ments (Thermo Fisher Scientific) were then added in DMEMfor 10 min. After washing away the excessive latex beads,RAW264.7 macrophages were incubated for a further 0 – 80min. Cells were homogenized, and LBP were purified as inBecken et al. (28) but by placing the density gradient in a 2-mlminicentrifuge tube that was centrifuged for 30 min and 1250 �g at 4 °C in a swing out table top centrifuge rotor. Colocalizationbetween BSA-rhodamine and latex beads was assessed afterfixation and mounting on glass slides using an Axioplan micro-scope (Zeiss).

Electron Microscopic Analysis—1 � 106 bone marrow-de-rived macrophages were labeled with ferrous nanoparticles (10nm) for 30 min followed by 3 h of incubation in fresh medium toensure labeling of lysosomes only. 1-�m murine IgG-Fc frag-ment-coated latex beads were added for 10 min (multiplicity ofinfection � 13) followed by 3 rinses of the cells to remove non-ingested latex beads. Cells were incubated for 10 min or 120min at 37 °C in fresh medium to allow phagosome maturationand then fixed in 2% (v/v) glutaraldehyde in 200 mM HEPESbuffer. For epoxy resin embedding, cell pellets were washedwith water, fixed with 2% (w/v) OsO4 (Electron MicroscopySciences) containing 1.5% (w/v) potassium ferricyanide, andblock-stained with 1.5% (w/v) uranyl acetate (Fluka) for 30 min.The cells were then dehydrated using a graded ethanol seriesand embedded in epoxy resin (Sigma). 70-nm-thin sectionswere cut on a Leica ultramicrotome Ultracut EM UCT (LeicaMicrosystems) using a diamond knife (Diatome) and stainedwith 0.2% (w/v) lead citrate (Taab) in 0.1 N NaOH for 10 s.Sections were analyzed with a CM100 transmission electronmicroscope (FEI). The images were recorded digitally with aQuemesa TEM CCD camera (Olympus Soft Imaging Solutions)and iTEM software v 5.1 (Olympus Soft Imaging Solutions).

The extent of the phagosome-lysosome fusion was catego-rized into arbitrarily defined classes of small, medium, and largevolume transfer. Membrane deposits that contained �20 fer-rofluid particles within a small and usually a pointed membraneprofile protruding from the phagosome membrane weredefined as “small.” “Medium” deposits were defined as contain-ing �20 ferrofluid particles and an area of protrusion �1⁄3 theproportion of the latex bead. “Large” deposits of ferrofluid con-tained �20 ferrofluid particles, and the protrusions exceeded 1⁄3of the latex bead area. The surface fraction of the phagosomemembrane over small deposits of ferrofluid was analyzed fromprofiles of at least 20 phagosomes on isotropic sections persample. Intersections of the phagosome membrane profile withthe horizontal and vertical lines of a lined test grid werecounted, and fractions were calculated from the total countsper sample.

Analysis of in Vitro Fusion between Latex Bead-containingPhagosomes and Lysosomes

Cell-free fusion of LBP with lysosomes was performed as pre-viously described (28) with modifications. Lysosomes and LBPwere isolated from RAW264.7 macrophages, and cytosol wasprepared from RAW264.7. Where indicated, isolated organ-elles were preincubated for 10 min at 4 °C with 10 �M SaliPhe orDMSO before the addition of the remaining components of the



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in vitro fusion reaction (final concentrations: 1� ATP-regener-ating system, 1� salt solution, 1 mM dithiothreitol, 2 mg/mlRAW264.7 cytosol). 10 �M SaliPhe or DMSO were present dur-ing the whole fusion reaction. After 30 min at 37 °C, the sampleswere set on ice for 5 min and incubated with 0.2 mg/ml protein-ase K (Qiagen) for 15 min. Then 1.75 mg/ml phenylmethane-sulfonyl fluoride was added and the volume increased to 200 �lwith HB. Latex bead phagosomes were isolated from the sam-ples, placing them on top of a 1-ml HB/25% (w/v) sucrose cush-ion in a swing-out rotor (1800 � g, 30 min). The collected pha-gosomes were diluted in HB and added to coverslips in 24-wellplates followed by centrifugation (690 � g, 15 min, 4 °C) andfixation (3% (v/v) formaldehyde, 2.5% (v/v) glutaraldehyde inHB) overnight. Remaining aldehyde was quenched with 0.1mg/ml NaBH4 for 30 min at room temperature, cells wererinsed with PBS, and coverslips were mounted in Mowiol(Sigma). Colocalization rates of latex bead-containing phago-somes with lysosomal BSA-rhodamine were determined fromat least 600 phagosomes per sample using fluorescence micros-copy (Axioplan).

Bacterial Killing

Bone marrow-derived macrophages were infected at a mul-tiplicity of infection of 5 with Escherichia coli DH5a or Listeriainnocua serovar 6a (NCTC 11288) in serum-free DMEM for 15min at 37 °C. After rinsing cells 3 times with PBS to removeexternal bacteria, DMEM containing 10 �g/ml gentamicin(Roth) was added to kill the remaining extracellular bacteria.Macrophages were lysed at 0, 20, or 60 min after infection with0.1% (v/v) Triton X-100, and serial dilutions were plated onnutrient agar plates to quantify colony forming units the nextday. Numbers of colony forming units at 0 min were set as100%.

Maturation of Rhodococcus Equi-containing Phagosomes

Heat-killed (15 min, 85 °C) R. equi 103� (31) were surface-labeled with TAMRA-S.E. (5-(and 6-)carboxytetramethylrhod-amine, succinimidyl ester; Life Technologies) at 0.1 �g/ml for30 min on ice, washed, and used for infection of J774E macro-phages seeded onto coverslips at an multiplicity of infection of10. Samples were centrifuged immediately for 5 min at 15 °Cand 200 � g. Medium was replaced by fresh, prewarmed com-plete medium containing either DMSO at 0.2% (carrier control)or 40 nM bafilomycin A1, and macrophages were placed at37 °C, 10% CO2. Cells were fixed after 2, 20, 60, and 120 min andprepared for immunofluorescence. Samples were stained withanti-transferrin receptor (TfR), anti-LAMP-1, or anti-EEA1and corresponding secondary Alexa Fluor 488-labeled antibod-ies. Samples were analyzed using a laser scanning microscopeLSM510 (Zeiss), and percentages of phagosomes colocalizingwith respective proteins were determined.

Statistical Analysis

All values are expressed as the mean � S.D./S.E. and analyzedvia two-tailed, unpaired Student’s t tests or one-way analysis ofvariance followed by a Tukey-Kramer test using GraphPadInstat 3 software (*, p � 0.05; **, p � 0.01).

Results

V0 Subunit a3 Deficiency Despite Normal pH of the Lysosome—In a genetic-based approach to analyze the role of the v-ATPase sub-unit a3 in membrane fusion, we used cells from Tcirg1oc/oc mice,which are homozygous for the osteosclerosis (oc/oc) mutation andnaturally lack the transmembrane a3-subunit of the v-ATPase (24).This subunit is particularly relevant here because in yeast the a-sub-unit has been previously implicated in the terminal phase of mem-brane fusion (11, 12) and the a3 isoform is the relevant a-isoform inmacrophage phagosomes and lysosomes (32). The a3-deficient oc/ocmice suffer from severe osteopetrosis and die early (24, 33) so that thepreferred cell type, primary macrophages, could not be obtained.However, we succeeded in generating fibroblast cell lines from thesemice, and we confirmed their lack of subunit a3-expression by West-ernblotting(Fig.1A),quantitativereal-timePCR(Fig.1B),andimmu-nostaining (Fig. 1C). The mRNA and protein concentration of thesubunita1was increased inthea3-deficientcells,whereas thesubunita2 shows unaltered expression level. Subunit a4 was only expressed ata low rate when compared with the other three isoforms in wild-typecells, and a4-mRNA concentration was not affected upon deletion ofsubunit a3 (Fig. 1, A and B). Concentrations of the V0 subunits d1 andc as well as the V1 subunit B2 remained unchanged (Fig. 1A). Surpris-ingly, fluororatiometric pH determination using Oregon Green 514demonstrated no difference in lysosome pH in a3-deficient versuswild-typecells(Fig.1,DandE),andacidification,visualizedwithLyso-Tracker Red staining, was highly sensitive to bafilomycin A1 treat-ment (Fig. 1, F and G) and, hence, dependent on a functionalv-ATPase complex.

Phagosome-Lysosome Fusion Occurs in the Absence of V0 Sub-unit a3—MEFs are non-professional phagocyte fibroblast cells.To study phagosome maturation, wild-type and oc/oc MEFswere stably transfected with an Myc-tagged CD32-cDNA cod-ing for the human IgG receptor Fc�RII (Fig. 2A), which medi-ates phagocytosis of IgG-coated particles. Cells were incubatedfor 15 min with IgG-coated latex beads, and maturation of theLBP was monitored over time by determining the colocaliza-tion of the internalized beads with the EEA1 and with late endo-somal/lysosomal LAMP-2 (Fig. 2B). Immediately after uptake,most beads colocalized with the early endosome marker EEA1in both genotypes (Fig. 2, B and C). Co-localization of the lyso-some membrane protein LAMP-2 with LBP increased steadilyand was observed with almost all phagosomes at 60 min ofchase. Kinetics of EEA1 loss from, and LAMP-2 acquisition toLBP were identical between wild-type and a3-deficient cells,indicating normal phagosome maturation in either case (Fig. 2,B and C).

Lysosome Alkalization Does Not Affect Maturation ofPhagosomes—To study if lysosome acidification per se, ratherthan the absence of the v-ATPase, modulates phagosome mat-uration, parallel samples were analyzed in the presence of thev-ATPase inhibitor bafilomycin A1, a non-covalent inhibitor ofthe proton pump, and nigericin, a fast-acting, membrane-per-meant K�/H�-exchanger. Concentrations were used that weresufficient for a complete loss of lysosome acidification (Fig. 1, Fand G). Neither pH modulator led to a significant difference inthe degree or timing of colocalization of early and late endocyticmarker proteins with the latex beads (Fig. 2D). This observation



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suggested a mechanism of phagosome maturation that is inde-pendent of luminal pH.

It is also of note that dextran (Fig. 3A) and transferrin (datanot shown) were taken up to the same extent by both wild-typeand mutant cells. Moreover, lysosome hydrolase activities (Fig.3B) and processing of cathepsin-D and cathepsin-L (Fig. 3C)also did not differ between a3-deficient and wild-type MEF cellsin steady state. These experiments indicate that fusion eventswithin the endocytic pathway are not affected by the lack of thea3 subunit.

Because we could not exclude possible compensatory pro-cesses for a3 by the subunit a1, which might mask acidification-independent functions, we further studied the consequences of

a deletion of all isoforms in professional phagocytes, such asmacrophages.

Disruption of v-ATPase Assembly after Conditional Knock-out of Atp6ap2—We took advantage of a recently generatedcKO mouse line in the gene for ATP6AP2. The encoded proteinis an accessory v-ATPase subunit and is also known as pro-renin receptor. This protein is a central factor in the assembly ofthe v-ATPase, and its removal results in the instability and dis-appearance of complete V0 complexes (21). The generatedAtp6ap2Flox/Flox Mx1-Cre cKO mice were treated for 1 weekwith polyinosinic-polycytidylic acid to induce Cre expression ininterferon �-responsive cells that include peritoneal macro-phages and stem cells that we used to induce BMDM. Macro-

FIGURE 1. The V0 subunit a3 is dispensable for lysosome acidification. MEFs generated from mice carrying the Tcirg1oc allele were analyzed for v-ATPaseexpression and their ability to acidify lysosomes. A, immunoblot analysis of v-ATPase subunit expression in whole cell lysates from wild-type (Tcirg1�/�) anda3�/� (Tcirg1oc/oc) MEFs. �-Actin staining was used to control for equal protein load. B, expression of the four subunit a isoforms was analyzed by quantitativereal-time-PCR in both genotypes. Shown are mean relative mRNA levels normalized to the most stable housekeeping genes �S.E. from three independentexperiments (**, p � 0.01, unpaired, two-tailed Student’s t test). n.d., not detectable. C, lysosomal localization of V0 subunit a3. Wild-type and mutant cells werefixed and co-immunostained with anti-V0 a3 and anti-LAMP-2 antibodies. Colocalization between v-ATPase subunit a3 and LAMP-2 was quantified to be 78%(Pearson’s correlation coefficient � 0.74) in wild-type cells and 15% (Pearson’s correlation coefficient � 0.09) in a3-deficient cells. D and E, ratiometricquantification of lysosome pH in wild-type and a3�/� MEFs. Late endocytic compartments of MEFs were pulse-chase labeled with dextran-Oregon Green 514,and cells were subjected to live cell fluorescence imaging. D, intensity ratios 440/488 as readouts for lysosome pH were calibrated in situ with nigericin atdefined pH. Additional control samples were preincubated for 15 min with bafilomycin A1 (B, 200 nM) or nigericin (N, 10 �g/ml, pH � 6.5 in both cases). E, thevalues for lysosome pH are comparable between a3-deficient and wild-type cells. Data are presented as the means � S.E. from three independent experiments.F and G, LysoTracker Red staining of MEF cells. Wild-type and a3-deficient MEFs were incubated with LysoTracker Red for 20 min and fixed for fluorescenceimaging. Where indicated, bafilomycin A1 (0 –500 nM) or nigericin (0 –20 �g/ml) was added for 15 min before LysoTracker Red staining as a control for alysosome pH equilibrated with the cytosol and extracellular buffer, respectively. Representative fluorescence images are shown in F. G, LysoTracker Red signalintensities were measured and plotted against the concentrations of bafilomycin A1 or nigericin to create dose-response curves. Shown are mean LysoTrackerRed signal intensities relative to carrier-treated controls cells �S.D. of three independent experiments. Scale bars � 10 �m.



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phages were analyzed 10 –17 days after induction and differen-tiation to BMDMs. This revealed that a deficiency of ATP6AP2could indeed be obtained and that the concentrations of severalV0 subunits, including subunit a isoforms 1–3, were signifi-cantly reduced in the cKO macrophages (Fig. 4A). However,loss of ATP6AP2 did not affect mRNA expression of these andadditionally tested subunits (Fig. 4B), suggesting posttranscrip-tional events leading to the loss of V0 subunits. Subunit a3showed the highest mRNA expression level of the four a-iso-forms, whereas a3 protein was almost completely missing, asdemonstrated by immunoblotting and immunofluorescencemicroscopy of differentiated macrophages (Fig. 4, A and C).Therefore, Atp6ap2 cKO is a suitable model to analyze an

almost complete absence (up to 96%) of V0 subunits of thev-ATPase.

In these cells the lysosome pH was only slightly less acidicthan in wild-type cells (Fig. 4, D–F), enabling us to study anpH-independent effect on lysosome fusion with phagosomes. Itis interesting to note that when treating cells with bafilomycinA1, ATP6AP2-deficient macrophages were somewhat moresusceptible to the drug than control cells (Fig. 4E), probably alsoreflecting the reduced number of v-ATPase complexes in theAtp6ap2 knock-out macrophages. As a measure of the biolog-ical relevance of v-ATPase in phagosome maturation, the kill-ing capacities toward bacteria of BMDMs from wild-type andinduced Atp6ap2 knock-out mice were compared (Fig. 4G).

FIGURE 2. Loss of V0 subunit a3 does not influence phagosome-lysosome fusion. A, total cell lysates of wild-type and a3-deficient MEFs, stably transfectedwith human Fc�RII-myc, were probed with the indicated antibodies in Western blot analysis. GAPDH was used to control for equal protein load. B, analysis oflatex bead phagocytosis in Fc�RII-myc expressing MEFs. Cells were pulsed with human IgG-opsonized latex beads for 15 min, and maturation of latexbead-containing phagosomes was monitored for chase periods from 0 to 60 min. External latex beads were labeled with an anti-hIgG antibody, and sampleswere fixed for immunostaining. Colocalization rates between internalized latex beads (no hIgG-signal) and EEA1 or LAMP-2 were determined by laser-scanningconfocal microscopy after the indicated chase periods. C, representative fluorescence images are shown for experiments conducted in B including magnifiedregions of interest. Arrows point to areas with colocalization (scale bars � 10 �m). DIC, differential interference contrast. hIgG, human IgG. D, influence ofmanipulation of luminal pH on phagosome maturation. Experiments in B were conducted in the presence of the v-ATPase inhibitor bafilomycin A1 (200 nM),the ionophore nigericin (10 �g/ml), or carrier control (DMSO) with the treatment starting either 15 min before (bafilomycin A1, DMSO) or concurrent with latexbeads incubation (nigericin). Percentages of phagosomes colocalizing with LAMP-2 and EEA-1 were determined by laser-scanning confocal microscopy. Aminimum of 100 phagosomes was analyzed per condition. Data are presented as the means � S.E. from 2–3 independent experiments.



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FIGURE 3. Endocytic maturation and degradation in lysosomes is normal in a3- and ATP6-deficient cells. Lysosome functions were analyzed in wild-typeand a3�/� MEFs and wild-type and Atp6ap2 cKO macrophages. A, visualization of vesicle maturation upon macropinocytosis and degradation within lyso-somes. Cells were incubated either in the presence of dextran-Texas Red (Dex-TR; 0.5 mg/ml) for 30 min plus a 2-h chase period or overnight in the presenceDQ-BSA (0.1 mg/ml). Samples were fixed and immunolabeled with anti-V0 a3 and anti-LAMP-2 antibodies. Distribution of the fluorescent marker proteins andtheir signal intensities were similar in the analyzed genotypes of both cell types. Shown are representative immunofluorescence images (scale bars � 10 �m).B, total �-hexosaminidase activity was comparable between a3-deficient and wild-type MEFs and between Atp6ap2 cKO and wild-type macrophages, respec-tively. Shown are the means � S.E. from 7–9 independent experiments. C, intracellular proteolytic processing of cathepsins D and L was analyzed by Westernblotting in the cell types mentioned in A and B. No impairment of enzyme maturation is visible. Arrows point to proform (P), intermediate (I), and mature (M)forms of the respective cathepsin. Lysates from cathepsin D (CtsD�/�) and cathepsin L (CtsL�/�) knock-out MEFs were used to control for antigen specificity ofthe antibodies (*, unspecific signal).



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Surprisingly, ATP6AP2-depleted macrophages killed E. coli(Gram-negative, avirulent) and L. innocua (Gram-positive,avirulent) rather more efficiently, suggesting that the absenceof the most of v-ATPase complexes did not affect their micro-bicidal functions. Compromising lysosome pH by applicationof bafilomycin A1 also did not change the killing capacity ofmacrophages toward E. coli (data not shown), implying bacte-ricidal mechanisms, which are independent on an acidic milieu.Similar to a3-deficient MEF cells, endocytic uptake and deliveryto lysosomes of dextran and BSA (Fig. 3A) also were notaffected in Atp6ap2 cKO macrophages; neither were matura-tion and activity of lysosomal hydrolases (Fig. 3, B and C).

Phagosome-Lysosome Fusion in ATP6AP2-depletedMacrophages—Knock-out of Atp6ap2 exclusively in cells ofthe myeloid lineage, including macrophages, due to LysM-me-diated Cre deletion similarly resulted in the disappearance ofthe a1- and a3-V0 subunits. Nevertheless in these cells we foundunaltered lysosome acidification and a significant colocaliza-tion of LAMP-2 with phagocytosed latex beads (Fig. 5), sugges-tive of unaltered phagosome maturation. To analyze at an ultra-structural level whether phagosome maturation was affected,we performed transmission electron microscopy experiments.The endocytic pathway of wild-type and induced Atp6ap2 cKOBMDMs was prelabeled with 10-nm ferroparticles and then

FIGURE 4. Killing of bacteria within macrophages is increased by a reduced v-ATPase concentration. Conditional knock-out of the v-ATPase accessoryprotein 2 (ATP6AP2) was induced in Atp6ap2Flox/Flox Mx1-Cretg/� mice by polyinosinic-polycytidylic acid treatment, and mice were sacrificed 10 days postinduction for isolation of peritoneal (A–F) or generation of BMDMs (G). Cells derived from Atp6ap2Flox/Flox Mx1-Cre�/� mice served as wild-type controls. A,Western blot analysis of v-ATPase expression. Cells were lysed and subjected to immunoblotting against v-ATPase subunits. Anti-ATP6AP2 antibodies to thefull length (fl), N-terminal (NTF), and C-terminal (CTF) fragments of ATP6AP2 were applied to determine Atp6ap2 knock-out efficiency. �-Actin was used tocontrol for equal protein load. B, mRNA expression levels of different v-ATPase subunits as determined by quantitative real-time-PCR. Values are presented inrelation to the most stable housekeeping genes as the means � S.E. from three (knock-out) to four (wild-type) macrophage preparations. n.d., not detectable.C, immunofluorescence analysis of localization of V0 subunit a3. Macrophages were fixed and labeled with anti-V0 a3 and anti-LAMP-2 antibodies. Colocaliza-tion between a3 and LAMP-2 was measured to be 72% (Pearson’s correlation coefficient � 0.78) in wild type and 24% (Pearson’s correlation coefficient � 0.27)in Atp6ap2 knock-out cells. D and E, primary macrophages were analyzed for lysosome acidification by LysoTracker Red staining. D, representative fluorescenceimages of macrophages either untreated or pretreated with bafilomycin A1 (100 nM) for 15 min. E, a dose-response curve was generated by measuringLysoTracker Red intensity after 15 min of pretreatment with various concentrations of bafilomycin A1 (0 –200 nM). Shown are mean intensities in relation toDMSO-treated samples �S.D. from two independent experiments. F, lysosome pH in Atp6ap2 cKO cells was determined using ratiofluorometric dextran-Oregon Green 514 measurements. Treatment for 1 h with bafilomycin A1 (200 nM) yielded a pH of �6.5 (not shown). Data are presented as the means � S.E.from four (wild-type) to six (knock-out) macrophage preparations. *, p � 0.05 according to unpaired two-tailed Student’s t test. G, killing of bacteria withinBMDMs. Macrophages were infected with either E. coli or L. innocua (multiplicity of infection � 5) (both non-pathogenic bacteria), and colony forming unitswere determined after the indicated infection periods by macrophage lysis and propagation on nutrient agar. Data are presented as percent of bacterialcolonies recovered from agar plates relative to the numbers recovered from 0-time samples. Shown are the means � S.E. from 2–5 independent experiments.*, p � 0.05 according to unpaired two-tailed Student’s t test between genotypes. Scale bars � 10 �m; insets show differential interference contrast images.



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incubated with 1.1 �m latex beads for 10 min followed byremoval of external beads and further incubation for 10 or 120min (Fig. 6A). Transmission electron microscopy visualizationof phagosomes (Fig. 6B) revealed three types of nanoparticle-containing fusion profiles between phagosomes and lysosomesthat were categorized as small, medium, and large (Fig. 6C). Asignificantly higher number of tight-fitting (small) membraneprofiles per bead was observed in Atp6ap2 cKO cells comparedwith wild-type cells. Medium and large profiles were observedat a much lower frequency in both genotypes with an even morereduced number in the Atp6ap2 knock-out cells compared withcells with wild-type v-ATPase (Fig. 6C). We used stereology tomake an estimate for the fraction of phagosome membrane thatsurrounds the small lysosomal ferroparticle deposits. It indi-cates the rate of small phagosome-lysosome fusion events andwas found to be significantly increased in ATP6AP2-deficientmacrophages at 10 min as well as 120 min of chase (Fig. 6D).Therefore, fusion frequency between LBP and lysosomes inmacrophages is, if anything, increased upon loss of a majorportion of v-ATPase.

Normal Phagosome-Lysosome Fusion after Arresting V0/V1Complexes—Our previous in vitro fusion data (28) and theabove latex bead experiments demonstrated that neither bafi-lomycin A1 nor nigericin inhibited phagosome-lysosomefusion. Similarly, bafilomycin A1 treatment, sufficient toincrease lysosome pH (Fig. 7A), did not alter maturation ofphagosomes containing heat-killed bacteria (31) within macro-phages (Fig. 7B). This suggested that vesicle acidification per se

is not a prerequisite for phagosome maturation. Recently, sali-cylihalamide, a v-ATPase inhibitor, was described to lock V0and V1 sectors of v-ATPases together (34). Because it has beenpostulated (11, 35) that the V0 subunit catalyzes membranefusion only when being free of V1, we investigated the V0-V1locking effect in vitro. The advantage of this drug is that boththe reduced availability of free V0 subunit and the effect ofincreased lysosome pH can be studied in one sample. Weobserved a clearly increased amount of assembled complexes inthe presence of the equipotent salicylihalamide analog saliphe-nylhalamide A (Fig. 8, A–C) as well as loss of LysoTracker Redsignals caused by an increased lysosome pH (Fig. 8D). Yet, therewas no effect on phagosome-lysosome fusion in vitro (Fig. 8E).Saliphenylhalamide A also did not change the degree of fusionof BSA-rhodamine-labeled lysosomes with latex bead-contain-ing phagosomes in macrophages (Fig. 8F), again questioning acentral role of v-ATPase participation in fusion.

Discussion

Does proton-pumping v-ATPase regulate and possibly evencatalyze phagosome-endosome and phagosome-lysosomefusion, and how does it participate in pathogen killing? Recentwork by other groups suggested that the V0 sector, the mem-brane integral parts of v-ATPases, acts downstream of SNAREcomplex formation and contributes to lipid mixing and mem-brane fusion (11, 35, 36). V0 sectors in only one of the two fusionpartners were reported to be sufficient to support fusion (35).The fusion of intermembrane fusion complexes appeared to

FIGURE 5. Knock-out of Atp6ap2 in LysM-Cre transgenic mice. Peritoneal macrophages harvested from Atp6ap2Flox/Flox LysM-Cretg/� (Atp6ap2 LysM-KO) andAtp6ap2Flox/Flox LysM-Cre�/� (control) mice were analyzed for v-ATPase expression and activity. A, expression of ATP6AP2 full length (fl) and C-terminalfragment (CTF) as well as expression of designated v-ATPase subunits was analyzed by immunoblotting of cell lysates from macrophages. �-Actin was used tocontrol for equal protein load. B, quantification of lysosome pH with ratiometric dextran-Oregon Green 514 measurements. Data are presented as the means �S.E. from five macrophage preparations for each genotype. C, uptake and intracellular delivery of latex beads. Atp6ap2 LysM-KO and control cells wereincubated with murine IgG (mIgG)-opsonized latex beads for 15 min and incubated for a further 60 min after washout of bead excess. Before fixation of the cellsfor immunofluorescence analysis, external latex beads were labeled with an anti-mIgG antibody (1 min). Lysosomal structures are visualized with an anti-LAMP-2 antibody. An anti-V0 a3 staining was performed to control for Atp6ap2 knock-out. Scale bars � 10 �m. DIC, differential interference contrast.



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require calcium release but was independent of the protonpumping activity of the v-ATPase (12, 37). We have chosenpharmacological and genetic approaches to analyze the contri-bution of the v-ATPase complex to fusion between phago-somes and endo(lyso)somes, which contain large quantities ofv-ATPase. In summary, surprisingly, neither of the abovev-ATPase manipulations yielded a significant change in theendocytic or phagocytic uptake, in lysosome acidification, or inphagosome-lysosome fusion. Our data suggest that neither

v-ATPase in general nor the a3 subunit in particular have anapparent direct role as a promoter of phagosome-lysosomefusion in our systems.

The role of the v-ATPase complex and specifically the mem-brane-bound V0 sector as a fusion-enhancing factor has been amatter of extensive investigations (13, 38). With respect to thephagocytic pathway, this role has only been studied by lookingat apoptotic body removal in zebrafish brain (16) where thea1-subunit of v-ATPase, despite an unaltered lysosome pH, was

FIGURE 6. Knock-out of Atp6ap2 enhances fusion between phagosomes and lysosomes. Fusion between phagosomes and lysosomes was analyzed inBMDMs from Atp6ap2 knock-out (Atp6ap2Flox/Flox Mx1-Cretg/�) and control mice by electron microscopy. A, scheme showing experimental procedures: lyso-somes are in red, and phagosomes are in orange: BMDMs were incubated with ferrous nanoparticles (10 nm, in black) and cultured in the absence ofnanoparticles for a further 3 h to ensure lysosomal localization of the tracer. Prelabeled macrophages were then fed with latex beads for 10 min at 37 °C.Phagosome maturation was stopped 10 min or 2 h later, and samples were processed for electron microscopy. B, representative micrographs show fusionprofiles between LBPs and nanoparticle-labeled lysosomes (Ly). Scale bars � 500 nm. Nanoparticle deposits within the phagosome (indicated by arrows) wereconsidered to reflect a recent fusion event. C, fusion events as depicted in B were then categorized according to their size, and their average number per beadwas compared between wild-type and Atp6ap2 knock-out cells after 2 h of chase. Atp6ap2 knock-out macrophages show an increase in the total number offusion events, reflected by an increase in the number of small deposits as the major population. LB, latex bead. D, the fraction of phagosome membranesurrounding the small deposits as a measure of the fusion rate was quantified by stereology and compared between both genotypes for at least 20 phago-somes per sample after 10 min and 2 h of chase. Data in C and D represent the means � S.E. from 4 (wild-type) to 7 (knock-out) macrophage preparations. *, p �0.05; **, p � 0.01 according to unpaired two-tailed Student’s t test.



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critical for fusion. This suggests that different phagocytic car-gos with different degrees of danger signals, e.g. apoptotic blebsversus IgG-opsonized latex bead phagosomes or bacteria, mayfeed into different maturation pathways. Also, the use of differ-ent organisms and tissues, zebrafish brain versus mouse macro-phage, and of different strategies for gene inactivation, mor-pholino addition versus conditional knock-out, may havecaused this. Another suggested correlation between v-ATPasepossession and phagosome maturation comes from phago-somes containing Mycobacterium tuberculosis. These phago-somes almost completely lack v-ATPase, and they are arrestedat an early stage along the endocytic/phagocytic continuum(39). This correlation suggested that the lack of the protonpump may not only be indicative for altered phagosome traf-ficking of this pathogen-containing phagosome but may actu-ally cause it.

Our approach to study proton-pumping-independent con-tributions of v-ATPases to membrane fusion was the genera-tion of cell lines disturbed in v-ATPase complex assembly thatcontain fewer V0 complexes (62–96% reduction varyingbetween different subunits), likely due to increased protein deg-radation. Furthermore, we have used cells with a knock-out ofthe a3 subunit. Both types of experiments challenged the cellu-lar functions of the transmembrane V0 sector (7). Of the fourpossible isoforms of the v-ATPase a-subunit, a3 is arguably themost important one on macrophage lysosomes and may be rel-evant for phagosome acidification (32). It was suggested thatthe a3 subunit is likely the most prominent in J774 andRAW264.7 macrophage cell lines (40, 41) in which it is localizedto phagosomes and needed for bactericidal function (32).a3-knock-out mouse embryonic fibroblasts have normal endo-cytic functions and unaltered activities of lysosomal hydrolasesat a normal lysosome pH. The complete lack of a3 also did notchange the kinetics of phagosome maturation, strongly sug-gesting that fusion of late endosomes/lysosomes with phago-somes occurs to the same extent in the absence of this a-subunitof the v-ATPase. It may well be that at least a portion of the a3subunit is replaced by the a1-subunit of v-ATPase as suggested

by the increased expression of this subunit in the a3-deficientMEF cells. Expression levels of other subunit a-isoforms wereunaltered or very low in protein and mRNA levels, making acompensatory role of these subunits unlikely. A limited func-tional compensation for subunit a3 by subunit a1 has been dis-cussed in osteoclasts, a cell population with predominantexpression of subunit a3 and only low levels of subunit a1 (42).Knock-out of a3 did not suffice to completely block the oste-oclast’s v-ATPase function (43).

Our studies using bone marrow-derived macrophagesfrom mice deleted in Atp6ap2, a gene essential for vacuolarATPase assembly, revealed that deletion resulted in a signif-icant drop in the concentrations of all v-ATPase V0 subunits.Similar to the situation in cardiomyocytes and kidney (21,44, 45), the presence of ATP6AP2 appears to be a prerequi-site for v-ATPase assembly in the endoplasmic reticulum.Taking the pivotal role of the v-ATPase complex intoaccount, it is not surprising that a complete deficiency ofv-ATPase may lead to early cell death (46). We expected thatthe pronounced loss of the v-ATPase levels would cause botha strong reduction in lysosome acidification and in mem-brane fusion between late endocytic organelles. Surprisingly,the ability to acidify these compartments was only slightly, ifat all, affected in the absence of functional v-ATPase. Thiscan possibly be explained by the presence of some v-ATPaseholo-complexes, which may also be a prerequisite for thesurvival of the knockdown cells. The expression levels of alla- isoforms, except for the isoform a4, which is not expressedin macrophages, were significantly lower in Atp6ap2 knock-out than in wild-type macrophages. It is likely that theremaining v-ATPase complexes are sufficient to fulfill theirproton-pumping function, as acidification was still bafilo-mycin A1-sensitive. However, less bafilomycin A1 is neededto increase the pH in Atp6ap2 knock-out cells, suggesting alower number of functional v-ATPase complexes.

The presence of a normally low lysosome pH allowed us tomonitor the fusion capacity of lysosomes with phagosomeswithout the need to consider abnormal acidification as a reason

FIGURE 7. Lack of an effect of bafilomycin A1 on maturation of phagosomes containing heat-killed bacteria. A, effect of bafilomycin A1 on lysosome pHin J774E macrophages. Cells were treated for 40 min with 10 nM bafilomycin A1 and then subjected to LysoTracker Red staining. Representative fluorescenceimages are shown (differential interference contrast channel in insets). Scale bars � 10 �m. B, influence of increased lysosome pH after bafilomycin A1treatment on maturation of R. equi-containing phagosomes. J774E macrophages were fed with heat-killed R. equi 103� and further incubated in the presenceof 40 nM bafilomycin A1 or 0.2% (v/v) DMSO (carrier control). Cells were fixed after the indicated incubation periods and prepared for immunofluorescenceanalysis by staining for EEA1, LAMP-1, or transferrin receptor (TfR). At least 100 phagosomes were analyzed for each condition. Data represent colocalizationrates between phagosomes and the respective marker protein as means � S.D. from 2–5 independent experiments.



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for any of the phenotypes we would observe. After all, distur-bances of trafficking events in the endocytic pathway caused bypH neutralization have been described (5). Despite the signifi-cant drop in the number of v-ATPase complexes in Atp6ap2knock-out cells, all tested functions of endocytic and phago-cytic compartments were unaffected, and phagosome-lyso-

some fusion as well as killing of bacteria was even somewhatpronounced. We expected that the large decrease in complexconcentration would affect the number of fusion events. How-ever, no effect on fusion was observed. Hence, the residual lev-els of v-ATPase complexes may still contribute to membranefusion.

FIGURE 8. Increased formation of V0/V1 complexes does not affect phagosome-lysosome fusion. Influence of the v-ATPase inhibitor SaliPhe on in vitro andin cellulo fusion between phagosomes and lysosomes was analyzed in RAW264.7 macrophages. A–C, effect of SaliPhe on v-ATPase complex formation. A,subcellular fractionation of RAW264.7 cells after 2 h of treatment with 10 �M SaliPhe or DMSO as indicated. T, total lysate; S1, low speed supernatant/perinuclearsupernatant; P1, low speed pellet/cells and nuclei; S2, high speed supernatant/cytosolic proteins; P2, high speed pellet/membrane bound proteins. SaliPhetreatment shifts v-ATPase V1 subunits B und E1 from the cytosolic to the membrane-bound fraction. LAMP-1 and V0c localization were not influenced bySaliPhe. Shown are representative immunoblots from two independent experiments (*, unspecific signal). B, SaliPhe increases V0-V1 interaction. Immunopre-cipitates (anti-V1B2) were prepared from cells treated as in A and analyzed for co-precipitation of V0 subunits. LAMP-1 was included as the negative control forinteraction. Representative blots from two independent experiments are shown. IP, immunoprecipitation. C, lysosomes from RAW264.7 cells were co-labeledwith BSA-rhodamine and nanomagnets and segregated via magnetic sorting. Isolated organelles were then incubated for 60 min under in vitro fusionconditions and processed for Western blotting. Where denoted, isolated lysosomes were pretreated for 10 min with SaliPhe (10 �M), and the treatment wascontinued throughout the experiment. Staining with the indicated antibodies shows more V1 bound to lysosomes when SaliPhe was present during theincubation. D, lysosome pH is increased by SaliPhe treatment. RAW264.7 cells were treated with 200 nM bafilomycin A1 or 10 �M SaliPhe for 100 min beforeLysoTracker Red staining. Shown are representative fluorescence images (insets: differential interference contrast channel). Scale bar � 10 �m. E, in vitro fusionexperiments were carried out with lysosomes treated as described in C, and LBPs were isolated from macrophages. Both organelle types were either pretreatedfor 10 min with SaliPhe or left untreated. In vitro fusion was then allowed to proceed for 60 min at 37 °C in the presence or absence of SaliPhe or carrier (DMSO).Incubation at 4 °C served as a negative control. F, influence of SaliPhe on in cellulo fusion of BSA-rhodamine-labeled lysosomes with LBPs. RAW264.7 macro-phages were cultivated in the presence of BSA-rhodamine overnight, and the label was chased into lysosomes for 2 h while incubating with SaliPhe or DMSO.Macrophages were then allowed to ingest latex beads for 10 min and incubated for a further 0, 20, or 80 min with the addition of SaliPhe/DMSO. Cells werelysed, and phagosomes were isolated to analyze the frequency of colocalization between BSA-rhodamine and LBPs. Data are presented as the means � S.E.from three independent experiments.



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The removal of the bulky V0 transmembrane protein com-plex (47, 48) from membranes might decrease steric hindrancefor membrane fusion catalyzed by other factors such as theSNARE complexes, thereby increasing fusion rates. In this con-text it is interesting to note that the size of protrusion of theyeast V0/V1 complex from the membrane has been estimated tobe 415 nm (49). The assumption that this huge complex mightimpair the contact between membranes also finds support inour electron microscopy-based observation of more fusionevents in the Atp6ap2-deleted macrophages as compared withthe wild-type cells.

This interpretation is in contrast to studies in yeast andmammalian cells that implicated a direct V0 sector involvementin fusion that is independent of the intraluminal pH (11–13, 16,37). However, some of this work is disputed, as a recent study onhomotypic vacuole fusion in yeast argues strongly that acidifi-cation rather than the presence of the v-ATPase is a majordriving factor of membrane fusion (17).

Contrary to these findings, in our experiments, acidificationalso did not seem to be a driving force, as neither nigericin norbafilomycin A1 treatment significantly altered the kinetics ofLAMP-2 acquisition by phagosomes containing latex beads orheat-killed bacteria. These observations suggest efficient pha-gosome-lysosome fusion under conditions of increased lyso-some pH. Also, locking many of the V0-V1 sectors together bysaliphenylhalamide A did not affect the extent of phagosome-lysosome fusion, although it could have been expected that thislocking would reduce the supply of free V0 and place extra bulkyprotein complexes between the two membranes destined tofuse (35).

Taken together, our data with cells either mostly lacking thev-ATPase complex or completely lacking its a3 subunit did notreveal an absolute necessity of the v-ATPase in fusion. Stronglysupportive of these findings is the fact that reconstituted fusionwith purified components of yeast vacuoles proceeded in thecomplete absence of a v-ATPase complex (50). The same wastrue for reconstituted homotypic early endosome fusion (51).Another piece of data pointing to a non-mandatory role forv-ATPases in membrane fusion is that compartments that nat-urally lack a v-ATPase complex, such as postmitotic nuclearvesicles (52), can still fuse. Although the above arguments anddata do not unequivocally exclude a regulatory role ofv-ATPases in fusion (53), they argue that the presence of thev-ATPase is not obligatory and that acidification-independentroles of v-ATPases in fusion may rather be the exception thanthe rule.

Acknowledgments—We thank Kristin Schröder, Christine Desel,Sönke Rudnik, and Janna Schneppenheim for technical support. Weare also very grateful to John Lucocq for help with stereology. Wethank the electron microscopy laboratory at the Department of Bio-science, University of Oslo, Norway.

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Brabander, Albert Haas and Paul SaftigBargen, Gareth Griffiths, Atsuhiro Ichihara, Beth S. Lee, Michael Schwake, Jef De

Sandra Kissing, Christina Hermsen, Urska Repnik, Cecilie Kåsi Nesset, Kristine vonVacuolar ATPase in Phagosome-Lysosome Fusion

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VacuolarATPaseinPhagosome-LysosomeFusiontein (DPGYDSIIYRMTNQ, Pineda-abservice). ExperimentalAnimals Mice with loxP sites flanking exon 2 of the Atp6ap2 gene have been described previously