A dual function of V0-ATPase a1 provides an endolysosomal degradation mechanism in Drosophila melanogaster photoreceptors

Abstract

The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity.

Figure 1.

A v100 mutant that rescues neurotransmission but causes cell death at high levels. (A) Alignment of 17 amino acids surrounding the arginine at position 755 (R755) shows 100% conservation across species. (B) Neurotransmitter release rescue experiments. Representative ERG traces from left to right show control (ctrl; yellow), v100-null mutant (eyFLP eye mosaic; blue), v100^R755A expression in WT photoreceptors (red), and v100^R755A expression in v100 mutant photoreceptors (green). Note that rescue with v100^R755A exhibits opposite phenotypes from the null mutant: rescue of the on transient (red circles) and a strong reduction of the response amplitude (depolarization). (C) Quantification of on transients for all four genotypes. (D) Quantification of depolarization for all four genotypes. v100^R755A in a WT background (red) and v100^R755A in a mutant background (green) compared with control (orange) and mutant (blue) are shown. (E–I) Eye pictures show dosage-dependent loss of photoreceptors upon v100^R755A expression in the mutant (H and I) but not upon v100^R755A expression in WT (G). Error bars indicate SEM. Asterisks denote statistical significance in pairwise comparisons with control (P < 0.001).

Figure 2.

V100 is required for the acidification of a subset of neuronal degradative compartments. (A–C’) Lysotracker live measurements in eye–brain cultures. (A and B) Representative scans of v100 MARCM eye discs (mutant cells marked with GFP). (A’ and B’) Only Lysotracker channel. (C) Live scan of a v100 MARCM eye expressing v100^R755A only in the mutant cells marked by GFP. (C’) Only Lysotracker channel. (D) Quantification of Lysotracker data in A–C’ shows a significant reduction in Lysotracker signal after photoreceptor differentiation at P + 40% (orange, control [ctrl]; blue, mutant). Expression of v100^R755A in the mutant (green) does not rescue the Lysotracker signal reduction. Gray, expression of v100^WT in mutant photoreceptors. (E) Identification of Lysotracker-positive compartments by live imaging using GFP-Rab5, GFP-Rab11, GFP-Rab7, and Atg8-GFP. Note that the ratio of Rab7/Lysotracker and Atg8/Lysotracker compartments is unaltered in the mutant despite the 50% Lysotracker reduction shown in D. The inset shows Rab7-positive late endosomes that are filled with Lysotracker (arrows). The arrowhead shows a Rab7-negative, lysotracker-positive ring, presumably a lysosomal or autophagosomal structure. (F–H) Block of all v-ATPase–dependent acidification using bafilomycin A1 in L3 larval eye discs with mutant v100 MARCM clones marked by GFP. Error bars indicate SEM. Bars: (A’ and F’) 10 µm; (E) 1 µm.

Figure 3.

V100 is predominantly an early endosomal protein in addition to its localization to synaptic vesicles. (A) Schematic of intracellular localization of markers and compartments in WT. (B) WT colocalization of 16 markers with V100 in developing photoreceptor terminals at ∼P + 25%. (C and D) Double labeling of a developing optic lobe for Syx7/Avl (purple) and V100 (green). (D) High resolution section of the developing lamina. Arrows in C indicate increased labeling of the lamina, which is shown at a higher resolution in D. (C’–D’’) Single channels from C and D are shown. (E) Triple labeling of a developing eye–brain section. Green, photoreceptor-specific expression of Rab5-GFP; blue, V100; red, Sunglasses/CD63. Note that V100 and Rab5-GFP colocalize at the developing photoreceptor terminals (lamina plexus), whereas Rab5-GFP is also enriched at the developing rhabdomeres in the cell bodies (eye disc). Blue arrows show colocalization of GFP-Rab5 and V100 at synapses, green arrowheads indicate absence of colocalization of GFP-Rab5 and V100 at the rhabdomeres, and red arrowheads indicate absence of colocalization of Sun and V100. (E’’’) In contrast to V100, the lysosomal marker Sunglasses marks different compartments, mostly in the cell bodies. EE, early endosome; MVB, multivesicular body/late endosome; Ly, Lysosome; SV, synaptic vesicle; RE, recycling endosome; RV, recycling vesicle. Error bars indicate SEM. Bars: (C) 10 µm; (D) 1 µm; (E) 5 µm.

Figure 4.

Loss of v100 causes endosomal and autophagic accumulations. (A) Schematic of heterogeneous intracellular accumulations in the v100 mutant. (B) Ratio of expression levels in mutant photoreceptor terminals compared with WT control (ctrl) in 50% mosaics. The red line represents unaltered levels. Note that numerous endosomal and lysosomal membrane markers are up-regulated. (C and D) Representative transmission electron micrographs reveal aberrant multivesicular structures and AVs (MVBs [arrowhead] and AVs [arrow]) in v100 mutant photoreceptor terminals. (E) Quantification of MVB-like (single membrane) and AV-like (double membrane) structures per photoreceptor terminals show a more than fivefold increase in the mutant (P < 0.01). (F and G) Coimmunolabeling of early endosomes using Syx7 and Rab5 reveals strong up-regulation in mutant cells. (H and I) Coimmunolabeling of Syx7 and Rab7. (J and K) Colabeling of autophagosomes using Atg8-GFP and anti-Syx7 labeling reveals both makers accumulating in mostly separate compartments. (L and M) Colabeling of early endosomes using 2xFYVE-GFP and lysosomes using anti-Sunglasses reveals that both markers accumulate in mutually exclusive compartments. EE, early endosome; MVB, multivesicular body/late endosome; Ly, Lysosome; SV, synaptic vesicle; RE, recycling endosome; RV, recycling vesicle. Error bars indicate SEM. Bars: (C) 5 µm; (G) 1 µm.

Figure 5.

V100 exerts an acidification-independent function on early endosomes. (A–C″) High resolution section of WT (A) and v100 mutant (B and C) 1-d adult photoreceptor terminals expressing pHluorin (green) and immunolabeled for CSP or Syx7/Avl (magenta). Note that pHluorin forms accumulations that exclude CSP (arrowheads) but partially colocalize with Syx7 (arrows) in the mutant. (D–F) Levels of pHluorin accumulation in live adult photoreceptor terminals at equal pH for the genotypes indicated. (G and G′) Negatively marked v100 mutant clone (magenta, WT terminals). Both mutant and control (ctrl) clones equally overexpress pHluorin and v100^R755A. Note that v100^R755A expression only in the mutant terminals leads to pHluorin accumulations. The dotted line approximates the clonal boundary. (H–M) Live confocal scans of P + 20% eye discs before and after 10-min incubation with the v-ATPase inhibitor bafilomycin 1A. (H and I) Control before and after bafilomycin treatment. (J and K) v100 mutant before and after bafilomycin. (L and M) v100 mutant with v100^R755A rescue. (N) Quantification of fluorescence increases after bafilomycin treatment shows that pHluorin accumulates in compartments that are acidified in a v-ATPase–dependent manner. Error bars indicate SEM. Bars, 10 µm.

Figure 6.

V100 interacts with the endosomal Syx7/Avl but not the Golgi/lysosomal Syx16. (A) Immunoblots of co-IP with anti-V100 antibody from adult fly head extract, probed with antibodies against Syx1A, Syx7/Avl, and Syx16. IgG, analogous co-IPs using preimmunoserum instead of anti-V100 serum. (B) Co-IP with anti-Syx7/Avl antibody from adult fly head extract probed with anti-V100. (C) Pull-down from adult fly head extract using bacterially made His-V100–N terminus. Immunoblot probed with anti-Syx1A, -Syx7, and -Syx16. (D) Pull-down from adult fly head extract using bacterially made His-Syx7. As negative control (His-CtrlProt), we used a bacterially made His fusion to a fly lipid enzyme (CG8630). Immunoblot probed with anti-V100. (E) GST pull-down of bacterially made His-Syx7 with bacterially made GST-V100–N terminus.

Figure 7.

Loss of v100 causes slow, adult-onset neurodegeneration. (A and B) ERG recordings of 1-wk-old flies. Note that mutant ERGs lack the on transient (arrows) but display normal depolarization. (C and D) Eyes from 1-wk-old WT and v100 mutant flies labeled with the photoreceptor-specific rhabdomere marker chaoptin (green) and the nuclear marker Toto-3 (blue). (E and F) ERG recordings from 2–3-wk-old flies show a slight reduction in depolarization. (G and H) 2–3-wk-old WT and v100 mutant eyes. (I–L) ERGs and immunolabeling of 5-wk-old WT and mutant eyes reveal progressive degeneration. (M–P) ERG recordings and immunolabelings from 5-wk-old flies kept in the dark show attenuated mutant phenotypes. (Q) Quantification for ERG depolarizations. Ctrl, control. Error bars indicate SEM. Bar, 10 µm.

Figure 8.

Selective rescue of the acidification-independent function accelerates degeneration by increasing accumulations in Syx7-positive, degradation-incompetent compartments. (A) Summary of V100 functions and v100^R755A rescue data. Our findings indicate two acidification-independent functions that are rescued by v100^R755A: synaptic vesicle (SV) fusion through Syx1A interaction and early endosomal (EE) fusion through Syx7 interaction. In contrast, V100 has a function as part of the V0V1 holoenzyme once endosomes mature into degradative compartments that are not rescued by v100^R755A. Consequently, selective rescue of the acidification-independent function leads to increased accumulations in Syx7-positive, degradation-incompetent compartments. (B, C, and D) 1-d adult photoreceptor terminals for control (B), v100-null mutant (C), and v100^R755A rescue in the v100 mutant (D). (B’, C’, and D’) Chaoptin alone (green channel) is shown. (B’’, C’’, and D’’) Syx7 alone (magenta channel) is shown. In the v100^R755A rescue, most chaoptin accumulates in large, Syx7-encircled MVB-like structures (arrows). RE, recycling endosome; RV, recycling vesicle. Bar, 5 µm.