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First published online 30 May 2006
doi: 10.1242/jcs.02983

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The SzA mutations of the B subunit of the Drosophila vacuolar H⁺ ATPase identify conserved residues essential for function in fly and yeast

¹ Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NU, UK
² Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NU, UK

View larger version (59K): [in a new window]   Fig. 1. Identification of point mutations in lethal alleles of Drosophila vha55. (A) Sample results from PCR-based SSCP gel, showing extra bands in mutants (arrows). C, control wild-type (Oregon R) genomic DNA; mutant strains are as described in the text. (B) Sequencing results. Point mutations and amino acid changes are indicated above the vha55 gene structure diagram. The four exons are labelled E1-E4; the introns are not to scale. The translated region is shown in black. Nucleotide numbers are relative to the Gadfly annotation for transcript A. Alignments for each of the mutated regions are shown: the mutated residue is underlined in the Drosophila sequence. The genes used are: D. melanogaster, vha55; H. sapiens, ATP6V1B2; S. cerevisiae, vma2; A. thaliana, At1g76030.

View larger version (61K): [in a new window]   Fig. 2. Heterozygous phenotypes and rescue of homozygotes in Drosophila epithelial transport assay. Three lines carrying recessive alleles of vha55 (A, vha557e1; B, vha5514; C, vha55SzA9) were out-crossed to wild-type flies, and fluid secretion rates by the heterozygotes (blue circles) were compared with wild-type tubules (black squares). Homozygous mutants that had been rescued by ubiquitous (heat-shock-GAL4 driven) expression of vha55::GFP were also tested (red diamonds). After resting rates were recorded, the tubules were maximally stimulated by the addition of the diuretic neuropeptides Capa-1 (Kean et al., 2002) and drosokinin (Terhzaz et al., 1999), both at 10-7 M, at 30 min. Data are expressed as mean ± s.e.m. (n=8). (D-F) The VHA55::GFP fusion protein localises correctly to the apical domain of Malpighian tubules in transgenic Drosophila. Immunocytochemistry of wild-type tubule with anti-VHA55 (D), negative control with primary antibody blocked with excess antigenic peptide (E) and direct epifluorescence view of a transgenic tubule with VHA55::GFP expression directed to the principal cells (F). The tubule is arranged as a simple epithelium, with wide, shallow cells wrapped around the central lumen. The apical localisation is established by the bulging of the V-ATPase fluorescence to the apical, rather than basal, side of the nuclei (visible as black holes in D&E, counterstained with DAPI in F). Bars, 10 µm. A confocal stack of the ICC is provided as Movie 1 in supplementary material.

View larger version (43K): [in a new window]   Fig. 3. Localisation of VHA55::GFP in Drosophila tissues. The VHA55::GFP fusion was driven ubiquitously using a heat-shock-GAL4 driver, and distribution in tissues monitored by fluorescence microscopy. (A) Salivary gland, showing prominent apical brush border, intercellular boundaries, perinuclear region and reticular network throughout the cell. (B) Higher-power view, showing reticular network clearly. (C) Anterior midgut: only the cuprophilic (`goblet') cells are labelled. (D) Hindgut: fluorescence appears in a broad region of the hindgut, and a ring around the anterior rectum, but the ion-transporting rectal pads (right) are conspicuously unstained. (E) Ovaries, showing localised labelling of nurse cells and immature oocytes. (F) Testes, with regional staining of the ejaculatory bulb. (G) In situ hybridisation to adult tubule with Rhodamine-labelled probe for vha55. This confirms localisation of the transcript to principal cells: a stellate cell (identifiable by its smaller nucleus) is conspicuously unstained. (Nuclei are labelled blue with DAPI.)

View larger version (72K): [in a new window]   Fig. 4. Drosophila vha55 complements vma2 in yeast. The Drosophila vha55 gene was cloned into in plasmid p415 met 25 (low copy, left), or p425 met 25 (high copy, right). 1, empty vector in wt yeast; 2, vma2 in wt yeast; 3, vha55 in wt yeast; 4, vha55::GFP construct in wt yeast; 5, vha55-ORF construct in wt yeast; 6, vha55::GFP in vma2 strain; 7, vha55-ORF in vma2; 8, vha55 in vma2; 9, VMA2 in vma2; 10, empty vector in vma2. (The upper panel plates were permissive: Leu-, Met-, pH 5.0; the lower panel plates were restrictive: Leu-, Met-, pH 7.5. Only yeast with functional V-ATPase can grow at pH 7.5.)

View larger version (35K): [in a new window]   Fig. 5. Effect of Drosophila vha55 point mutation residues in yeast. The point mutations identified in Fig. 1 were introduced into vha55::GFP in low-copy plasmids. Mutations of vha55 correspond with the different point mutations described in Fig. 1. The left panel plates were permissive; the right panel plates were restrictive.

View larger version (84K): [in a new window]   Fig. 6. Growth of yeast carrying vha55 mutants. (A-B) 1x106 cells were taken from mid-log phase culture, serially diluted 1:5 eight times, and each dilution spotted in a grid on both pH 5.0 (permissive, left panels), and pH 7.5 (restrictive, right panels) selective medium plates, and grown at 30°C for 2-3 days. (A) Low-copy plasmid. Column 1, wild-type, no plasmid; 2, vma2, no plasmid; 3, vma2 with VMA2 rescue; 4, vma2 with vha55 rescue; 5, vma2 with vha55::GFP rescue; 6, vma2 with vha55 ORF::GFP rescue. (B) As A, except that vma2 yeast were carrying vha55::GFP cDNA in a low-copy plasmid, with point mutations corresponding to the vha55 mutant alleles. Column 1, vha55::GFP cDNA; 2, empty vector; 3, vha55SzA1; 4, vha55SzA9; 5, vha55SzA9 (both changes); 6, vha55SzA12; 7, vha557e1; 8, vha5514.

View larger version (83K): [in a new window]   Fig. 7. Functional assay of vacuole acidification by V-ATPase. (A) Quinacrine staining of acidified vacuoles for vma2 strains carrying wild-type Drosophila vha55 constructs in low-copy plasmids. Upper panels show Quinacrine-stained cells under epifluorescence; lower panels are phase-contrast images. A bright vacuole indicates functional acidification; a dark vacuole indicates inactivation of the V-ATPase. The vacuolar acidification phenotype is thus rescued both by VMA2, and by all constructs encoding vha55. (B) As A, except that yeast were carrying vha55::GFP plasmids with mutations corresponding to the alleles shown. Although the yeast can survive under these permissive conditions, none of the constructs rescue the acidification phenotype. (Although the vacuole in e.g. vha557e1 can appear less dark, this is due to an out-of-focus contribution from the cytoplasm; the contrast with functional vacuoles in the top panel is clear.)

View larger version (15K): [in a new window]   Fig. 8. Rescue of V-ATPase activity by vha55 and VMA2 constructs. V-ATPase activities were measured as Vmax in yeast vacuole preparations. Black bars, wild-type, vma2 and rescued yeast; empty bars, vma2 yeast rescued with vha55 cDNA mutated with SzA defects. Data are mean ± s.e.m. of assays on three independent cultures, and are expressed as a percentage relative to wild-type yeast: the average Vmax of control preparations was 2.3±0.5 µmol Pi/minute/mg protein, comparable with values reported by others (Curtis et al., 2002; MacLeod et al., 1998).

View larger version (13K): [in a new window]   Fig. 9. Western analysis of mutant VHA55 expression levels in yeast. Mutant vma2 yeast were grown under selective (Met-), but pH-permissive (pH 5) conditions, and vacuoles harvested and analysed by western blot with anti-VHA55 antibody. Yeast contained plasmids containing either vha55::GFP, or vha55::GFP with the point mutations corresponding to those described in Fig. 1.

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