Generation of Atp6v0a2 mouse models

To generate the targeting vectors for the Atp6v0a2−/− and Atp6v0a2RQ/RQ mice a modified recombineering protocol was used [60]. Exons 10 to 20 of the murine Atp6v0a2 gene (NCBI38: Chr.5 124,711,748–124,723,441) were transferred from a bacmid (BMQ-337012, Sanger resources) to the pDTA vector (kindly provided by Carmen Birchmeier, Max-Delbrück-Center, Berlin) by homologous recombination, resulting in the genomic subclone. The neomycin cassette flanked by FRT and loxP sites was introduced between the exons 14 and 15 by homologous recombination between the pHW025 vector (kindly provided by Carmen Birchmeier, Max-Delbrück-Center, Berlin) and the genomic subclone. Utilizing cre recombinase the neomycin cassette was deleted, resulting in a single loxP site between exon 14 and 15. This vector was digested with EcoRV between Exon 17 and 18 and ligated with a digestion product of the pHW025 derived by EcoR1 and Ale1 and subsequent blunting (Fig. S1A). G4 embryonal stem cells were used for homologous recombination (Andras Nagy, MSHRI, Toronto) and aggregated with donor morulae. The resulting mouse line was crossed with germline cre recombinase expressing mice (Hprt-cre mice) giving rise to the Atp6v0a2−/− mouse line (Fig. S1B). For the generation of the Atp6v0a2RQ targeting vector a PCR product of the vector ranging from the neomycin cassette to the intron 18 was mutated within the exon 18 to generate the R755Q mutation. This PCR-fragment as well as the Atp6v0a2−/− targeting vector were digested with RsrII and Nde1 and ligated together (Fig. S1C). Mating of the resulting mice with germline flippase recombinase expressing mice (CMV-Flip mice) resulted in the Atp6v0a2RQ mouse line (Fig. S1D). For genotyping of the mice, PCR was performed as described (Fig. S2).

Resulting heterozygous Atp6v0a2 knock-out and knock-in mice were inbred to produce homozygous mice (Atp6v0a2−/−, Atp6v0a2RQ/RQ). Experiments were performed with mice in C57Bl/6 background and littermates were used as controls. Mice were housed in the animal facility of the Max Planck Institute for Molecular Genetics. 3R rules were followed in that the minimum numbers of animals were used for experiments and that the maximum number of experiments were performed in vitro. Permissions for animal breeding and experiments were obtained from the Landesamt für Gesundheit und Soziales (LAGeSo) in Berlin, Germany (No. G0346/13, G0176/19), and the Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES) in Oldenburg, Germany (No. 20/3483).

mRNA expression analysis

Tissue was snap frozen and crushed using a mortar. Total RNA was extracted from homogenized tissue with AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany). Total cDNA was reverse transcribed by RevertAid H Minus First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, USA). Quantitative PCR was performed using EVAgreen (Solis BioDyne, Tartu, Estonia) on a QuantStudio 3 Real-Time-PCR System (ThermoFisher, Waltham, USA). All primer sequences are available in Table S4.

Spermiogram analysis

Spermatozoa were freshly isolated from the cauda epididymis of adult mice and 5 min incubated in Human Tubal Fluid medium. Sperm motility and concentration were measured using IVOS sperm analyzer (Hamilton Thorne, Beverly, USA). Sperm were observed for 30 s and images were acquired at a frequency of 60 Hz. The cutoff of motile sperm was 30 μm/s for linear velocity.

Histology

Mice were deeply anaesthetized and transcardially perfused with 4% PFA and organs were sampled. Tissues for paraffin sectioning were fixed in 4% PFA at 4 °C overnight and sectioned at Microm HM 355 S (Microm, Walldorf, Germany) at 5 µm thickness. Brain samples were stained with kresyl violet. Testis, epididymis and skin samples were stained with hematoxylin/eosin.

Ultrastructural analysis

Skin samples were fixed for at least 2 h at room temperature in 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer pH 7.4, cut into pieces of ca. 1 mm3, washed in buffer, postfixed for 1 h in 1% aqueous osmium tetroxide, rinsed in water, dehydrated through graded ethanol solutions, transferred into propylene oxide, and embedded in epoxy resin (glycidether 100). Semithin and ultrathin sections were cut with an ultramicrotome (Reichert Ultracut E)(Leica Microsystems, Nussloch, Germany). Semithin sections of 1 µm were stained with methylene blue. 60–80 nm ultrathin sections were treated with uranyl acetate and lead citrate and examined with an electron microscope JEM 1400 equipped with a 2K TVIPS CCD Camera.

Testes were collected from adult mice and small pieces were fixed in 1.5% glutaraldehyde, 1.5% formaldehyde, 0.15 mol/L HEPES/KOH (pH 7.4) at 4 °C until embedding and sectioning. For epoxy resin embedding, samples were postfixed in 1% osmium tetroxide in aqua bidest, stained in half-saturated watery uranyl acetate, dehydrated in an ascending ethanol series and finally embedded in Agar 100 (Agar scientific Ltd., Stansted, UK). Ultrathin sections were cut using an ultramicrotome and examined by TEM (Zeiss EM 902, Oberkochen, Germany). Digital images were captured with a slow-scan 2 K CCD camera (TRS, Tröndle, Moorenweis, Germany).

Cell culture

Mouse embryonic fibroblasts (MEFs) were isolated from e13.5 embryos using trypsin–EDTA (Gibco, ThermoFisher, Waltham, USA) and cultivated in DMEM (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (Gibco, ThermoFisher, Waltham, USA), 1% UltraGlutamine (Lonza, Basel, Switzerland) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland). As positive controls, the wildtype cells were treated with monensin (20 µM), 2F-peracetyl-fucose (200 µM), chloroquine (50 µM), bafilomycin A1 (200 µM) or ammonium chloride (25 µM) (all from Sigma Aldrich, St. Louis, Missouri, USA). MEFs were immortalized with SV40 large T antigen transfection (Addgene plasmid #21826) using Lipofectamine 3000 Reagents according to manufactural instruction (ThermoFisher, Waltham, USA).

Viral overexpression of V0a2

For viral overexpression of the V0a2 subunit, the human ATP6V0A2 cDNA sequence tagged with V5 (GKPIPNPLLGLDST) was cloned into the vector pLXIN (Clontech Laboratories, California, USA) with or without the R755Q mutation. Using Phoenix ampho packaging cells (ATTC, Manassas, USA), retroviruses were produced to infect HeLa cells. Stable cell lines with mild overexpression were generated by selection with 0.6 mg/ml G418 (Gibco, ThermoFisher, Waltham, USA).

Immunostaining

Paraffin sections were deparaffinized, rehydrated and equilibrated in PBS. Antigen unmasking was performed using citric acid (pH 6.0) or pepsin antigen retriever (Sigma Aldrich, St. Louis, Missouri, USA) followed by permeabilisation and blocking in 0.2% Triton-X/5% BSA/5% goat or donkey serum. Antibodies used are as follows: Anti-laminin (#L9393, Sigma-Aldrich, St. Louis, USA), Anti-Ctip2 (#ab18465, Abcam, Danaher, Washington, USA), Anti-Tbr2 (#AB15894, MerckMillipore, Darmstadt, Germany), Anti-IIH6 α-Dag1 (#05–593, MerckMillipore, Darmstadt, Germany), Anti-ß-Dag1 (#7D11, Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242); Acrosome were stained with Lectin PNA conjugated with Alexa Fluor 568 (#L32458, Sigma-Aldrich, St. Louis, USA). Cultured cells were fixed with 4% PFA, permeabilized and blocked with 0.1% Saponin/ 3% BSA. The following antibodies were applied: Anti-GM130 (#610823, BD Biosciences, Heidelberg, Germany), anti-Calreticulin (#ab92516, Abcam, Danaher, Washington, USA), antiTGN46 (#AHP500GT, Bio-Rad, Feldkirchen, Germany), anti-GOPC (#12163-1-AP, Proteintech, Planegg-Martinsried, Germany), anti-eGFP (#ab6556, Abcam, Danaher, Washington, USA) and anti-V5 (#V8012, Sigma-Aldrich, St.Louis, USA). Sections and cells were decorated with secondary anti-IgG antibodies conjugated with Alexa Fluor 488 (#A21202, Invitrogen, ThermoFisher, Waltham, USA) and Alexa Flour 555 (#A21572, Invitrogen, ThermoFisher, Waltham, USA) or primary antibody was direct labeled with APEX antibody labeling kit (ThermoFisher, Waltham, USA). Fucose residues were stained with Lectin AAL (#B-1395, Vector Laboratories, Newark, USA) for 1 h and detected with streptavidin Alexa Fluor 555 conjugate (#S21381, Invitrogen, ThermoFisher, Waltham, USA). DNA was stained by DAPI (#D1306, Invitrogen, ThermoFisher, Waltham, USA). Sections and cells were mounted in FluoromountG (SouthernBiotech, Birmingham, USA). Fluorescence pictures were taken with LSM 700 (Zeiss, Jena, Germany), Celldiscoverer 7 (Zeiss, Jena, Germany) or BX60 microscope (Olympus, Tokyo, Japan). To facilitate detailed analyses of co-localization, separation of Golgi stacks was induced through treatment of cells for 3 h with 33 µM nocodazol (Sigma-Aldrich, St. Louis, USA).

Metabolic labeling of sialylated glycoproteins using ManAz

Cells were seeded on coverslips and preincubated with 50 µM Chloroquine (CQ), 200 mM Bafilomycin a1 (Baf), 2 µM Tunicamycin (TN). The next day, cells were pulsed 6 h with 50 µM tetraacetylated N-Azidoacetyl-mannosamine (ManNAz, Jena Bioscience, Jena, Germany). Cells were washed with PBS, fixed with 4% PFA and permeabilized 15 min with 0.5% Triton-X. Cells were labelled with click reaction solution (10 µM Fluor 488-Alkyne fluorescent probe/150 µM CuSO45H2O/300 µM BTTAA/2.5 mM Ascorbic acid/100 mM K2HPO4) for 1 h and nuclei were stained by DAPI. The fluorescence intensities of Golgi apparatus from individual cells were measured using Operetta High Content Imaging System (PerkinElmer, Rodgau, Germany). The experiment was performed three times.

Measurement of trans Golgi pH

The constructs coding for RpHLuorin2 fused to truncated GalT and GPI-anchored RpHLuorin2 have been described [27] and are deposited at Addgene (plasmid 171,719 and 171,721 respectively). MEF cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution (15240-062; Gibco, ThermoFisher, Waltham, USA). Cells were tested for mycoplasma contamination. MEF cells were transfected by electroporation with the plasmids using the NEON Transfection System (Invitrogen, ThermoFisher, Waltham, USA) with 10 μl NeonTips (MPK1025; ThermoFisher, Waltham, USA) using 1 pulse of 1350 V and 30 ms. The concentration of DNA used was 0.5 μg per 1 million cells. Cells were imaged 48 h post-transfection. Only cells expressing low to moderate levels of the transfected plasmids, based on fluorescence intensity and manual localization assessment, were chosen for subsequent microscopic analyses.

Imaging of cells took place in DMEM lacking phenol red, except for the pH calibration curve which was obtained in pH buffers composed of 125 mM KCl, 25 mM NaCl, and 25 mM N-[2-hydroxyethyl]-piperazine-N-[2-ethanesulfonic acid] (HEPES, pH 7.5, 7.0 and pH 8.0) or 25 mM 2-[N-morpholino] ethanesulfonic acid (MES, pH 6.5, 6.0, 5.5, 5.0 or 4.5). Each buffer solution was adjusted to the final pH using 1 M NaOH or 1 M HCl at 37 °C. For calibration with GPI-RpHLuorin2, the cells were preincubated in pH buffer for 15 min at 37 °C.

For microscopy, 50,000 MEF cells were seeded in 100 μl DMEM lacking phenol red and FBS in a four-compartment glass-bottom dish (627,870; Greiner, Kremsmünster, Austria). 4 h after transfection, the cell medium was refreshed with new DMEM medium containing FBS and cells were cultured overnight at 37 °C and 5% CO2. 48 h after transfection the medium was changed with imaging medium (DMEM without phenol red) 1 h before imaging. For the Bafilomycin A1 conditions, cells were treated with 200 nM Bafilomycin A1 (cat.code tlrl-baf1; InvivoGene) in DMSO (1st experiment) or 100 nM Bafilomycin A1 with 20 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP; second and third experiment) for 1 h prior to live imaging. Imaging was performed at 37 °C using a LSM800 Zeiss confocal laser scanning microscope with a 40 × 0.95 NA water immersion objective, using sequential excitation at 405 and 488 nm (Zeiss, Jena, Germany). Images were analyzed using FIJI-ImageJ as described [27].

Brefeldin A-induced golgi collapse

Cells were seeded on coverslips. The next day, cells were incubated with 5 µg/ml Brefeldin A (BFA) for 8 min and fixed in 4% PFA for 10 min. Cells were washed three times in PBS, permeabilized and blocked with 0.1% saponin in 3% BSA. Immunofluorescence staining was performed for Gm130 and Calreticulin. Pictures were taken with microscope BX60 (Olympus, Tokyo, Japan) and LSM 700 (Zeiss, Jena, Germany). At least 100 cells per sample were counted and the experiment was performed twice.

RUSH assay

Immortalized MEFs were seeded on coverslips. The next day, the cells were transfected with a Str-KDEL-IRES-TNFa-GFP-RUSH construct (according to [25, 61]) using Lipofectamine 3000 Reagents according to manufactural instruction (ThermoFisher, Waltham, USA). 24 h post transfection cells were incubated with 40 µM biotin (Roth, Karlsruhe, Germany) for 10, 15, 20 and 25 min and fixed in 4% PFA for 10 min. Cells were washed three times in PBS, permeabilized and blocked with 0.1% saponin in 3% BSA. Immunofluorescence staining was performed for GM130 and eGFP. Pictures were taken with Celldiscoverer 7 (Zeiss, Jena, Germany) and LSM 700 (Zeiss, Jena, Germany). At least 100 transfected cells per sample and timepoint were analyzed.

Flow cytometry analysis

Flow cytometry analysis was used to quantify an Aleuria Aurantia Lectin (AAL) surface staining on MEFs. Measurements were performed on a Cytoflex LX device (Beckman Coulter Genomics, Brea, USA) using 96-well U-bottom plates for the measurement. For the quantification, approximately 100,000 cells were transferred onto the 96-U-bottom-well plate and washed by adding 200 μL of PBS, centrifuging the plates at 400 g for 5 min at RT, discarding the supernatants, and resuspending the pellets in the remaining volume by vortexing briefly. For the individual staining procedure, a mastermix of the fluorophore conjugated lectin diluted in PBS was prepared. Twenty microliters of the mastermix were added per well. The plates were incubated for 15 min at 4 °C. The first staining step with Aleuria Aurantia Lectin (AAL), Fluorescein (FL-1391-1) (Vector Laboratories, Newark, USA) was performed with dilution (1:100). After incubation for 15 min, one washing step was performed prior to staining with DAPI. DAPI was diluted in PBS (1:100,000) before acquiring 50 µL. At least 600 cells/condition were analyzed. Data were analyzed using FlowJo_V10.

Membrane precipitation

Cells were rinsed with ice‐cold PBS, resuspended in homogenization buffer (20 mM HEPES (pH 7.5), 125 mM KCl, 50 mM sucrose, 1 mM EDTA, protease and phosphatase inhibitors), and homogenized by 12 strokes with a KIMBLE Dounce tissue grinder with a large clearance pestle (SigmaAldrich, St. Louis, USA). Then, nuclei were removed by centrifugation at 800 × g for 5 min and membranes pelleted from post-nuclear supernatants by centrifugation at 18,000 × g for 20 min. Resulting pellets were resuspended in lysis buffer (50 mM HEPES pH 7.4, 40 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1% Triton X‐100, 1 × Halt protease and phosphatase inhibitor cocktails).

Immunoblotting

Proteins were extracted with AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) and solved in 5% SDS RIPA buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% Triton X-100, 0.25% Desoxycholate, 5% SDS) supplemented with protease inhibitor (Complete, Roche, Mannheim, Germany). 15 to 20 μg of protein per lane were separated by SDS-PAGE and subsequently transferred to nitrocellulose membrane. After semidry blotting membranes were blocked 1 h with Blocking buffer (Li-Cor Biosciences, Lincoln, USA) and probed with primary antibodies. Antibodies were used as follows: Anti-Atp6v0a1 (#13828-1-AP, Proteintech, Planegg-Martinsried, Germany), Anti-Atp6v0a2 (#ab96803, Abcam, Danaher, Washington, USA), Anti-Atp6v0d1 (#ab202899, Abcam, Danaher, Washington, USA), Anti-Atp6v1a (#ab199326, Abcam, Danaher, Washington, USA), Anti-Atp6v1b2 (#14617S, Cell Signaling, Danvers, USA), Anti-Eln, Elastin Products Company), Anti-Dcn (#LF-114, Kerafast, Shirley, USA), Anti-Tgn38 (#ab16059, Abcam, Danaher, Washington, USA), Anti-Gpr56 (#MABN310, MerckMillipore, Darmstadt, Germany), Anti-Col3a (#C2C3, GeneTex, Irvine, USA), Anti-Gopc (#ab109119, Abcam, Danaher, Washington, USA), Anti-Gapdh (#AM4300, ThermoFisher, Waltham, USA) and Anti-ActB (#4970, Cell signaling, Danvers, USA). Lectins were produced by Vector Laboratories (Newark, USA): SNA (#B-1305), PNA (#B-1075), E-Pha (#B-1125-2), AAL (#B-1395). Membranes were washed and incubated with IRDye- HRPconjugated secondary antibodies. Lectins were incubated with IRDye-streptavidin. Total protein was stained using Revert™ 700 Total Protein Stain (LI-COR Biosciences, Bad Homburg, Germany). Signals were detected with OdysseyFc Imaging System and densitometric quantification was performed using Image Studio (LI-COR Biosciences, Bad Homburg, Germany). V-ATPase western blots were quantified using Image Lab software (Bio-Rad, Feldkirchen, Germany).

Proteome analysis

Confluent MEFs were washed twice with HBSS and once with DMEM w/o phenol red (Lonza, Basel, Switzerland). 6 mL DMEM w/o phenol red without FCS were added. After 24 h the supernatants were collected. The remaining cell layers were lysed in lysis buffer (3 M guanidinium chloride/5 mM Tris(2 carboxyethyl)phosphin, 20 mM chloroacetamide und 50 mM Tris(hydroxymethyl)aminomethan pH 8,5) and incubated at 96 °C for 10 min. Protein lysates were analyzed by LC–MS/MS and label-free quantification (LFQ) as previously described [62]. Data analysis was done with Perseus (v1.6.10.43). LFQ intensities, originating from at least two different peptides per protein group were transformed by log2. Proteins differentially expressed by at least one Log2 unit are given in Tables S1 and S2.

N-Glycan analysis

The proteins/glycoproteins were reduced and carboxyamidomethylated followed by sequential tryptic and peptide N-glycosidase F digestion and Sep-Pak purification. Permethylation of the freeze-dried glycans and MALDI-TOF-MS of permethylated glycans were performed as described elsewhere [63]. Three animals per genotype were analyzed.

Statistical analysis

One- and two-way analysis of variance (ANOVA), student’s t-test as well as Kruskal–Wallis test with appropriate multiple testing correction were performed using GraphPad prism 8 (v.8.3.0). Animal experiments were repeated at a minimum of three times.