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First published online 15 July 2008
doi: 10.1242/jcs.033084

Journal of Cell Science 121, 2612-2619 (2008)
Published by The Company of Biologists 2008
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Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa

Jonathan P. Day, Susan Wan, Adrian K. Allan, Laura Kean, Shireen A. Davies, Joe V. Gray and Julian A. T. Dow*

IBLS Division of Molecular Genetics, University of Glasgow, Glasgow, G11 6NU, UK


Figure 1
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  		Fig. 1. Distinct modalities for the plasma membrane V-ATPase. (A) On its own, a plasma membrane V-ATPase (V) can electrically polarise an apical domain to well over 100 mV. (B) When partnered with an apical chloride channel (typically a ClC), it is capable of bulk acidification. (C) When partnered with an apical alkali-metal/proton exchanger, it can drive active trans-epithelial transport of (for example) K+.

 

Figure 2
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  		Fig. 2. In situ hybridisation reveals epithelial expression of CG10806 and CG31052. (A) CG10806 anti-sense showing strong expression in the hindgut (HG) and weaker expression in the Malpighian tubules (MT), with weak staining in the midgut (MG). (B) CG31052 anti-sense showing expression in both the hindgut and the Malpighian tubules, with no staining in the midgut. (C,D) Negative controls; sense probes for CG10806 (C) and CG31052 (D) show no staining.

 

Figure 3
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  		Fig. 3. Although widely expressed, the three classical NHEs do not show the apical localisation required of the Wieczorek exchanger. Immunocytochemistry with antibodies raised against the indicated genes, using FITC or Texas Red secondary antibodies, is shown. Blue colour in some panels reflects nuclear staining with DAPI. Where not specified, tissues are from adults.

 

Figure 4
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  		Fig. 4. Immunocytochemical localisation of CPA2 family members to the apical plasma membrane in the Malpighian tubule. (A) Staining for endogenous CG10806 protein. (B) Staining for c42>UAS-CG10806-overexpressed protein. (C) Staining for endogenous CG31052 protein. (D) Staining for c42>UAS-CG31052-overexpressed protein. Insets show the cells corresponding to the asterisks, with nuclei labelled blue with DAPI and apical microvilli labelled green with phalloidin-FITC to demonstrate the apical location of the CPA2 proteins. (E) Negative control; no first antibody. Photographs were taken with the same exposure settings.

 

Figure 5
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  		Fig. 5. Both CG10806 and CG31052 localise to the apical plasma membrane. Flies transgenic for UAS-CG10806::eYFP or UAS-CG31052::eYFP fusions were crossed to GAL4 line c42 to drive expression in the Malpighian tubule principal cells, and subcellular localisation in 1-week-old adult progeny was established by confocal microscopy. DAPI was used to visualise the nuclei (blue) to demonstrate the apical localisation of the transgenic proteins. (A) CG10806 transcript A; (B) CG10806 transcript B; (C) CG31052. (D,E) For comparison, a vha55::GFP transgene marking the apical membrane (D) and immunocytochemistry against the Na+-K+ ATPase {alpha} subunit (a marker of the basolateral membrane), together with GFP-tagged vhaSFD to mark the apical membrane (E) are also shown. Lu, apical tubule lumen; AM, apical plasma membrane; BM, basal plasma membrane. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Complementation of salt sensitivity of the Saccharomyces cerevisiae {Delta}ena1-4, {Delta}nha1, {Delta}nhx1 mutant by CG10806 and CG31052. YFP-tagged CG31052 and CG10806 were cloned into the yeast expression vector pYes2.1, allowing expression of the recombinant protein upon galactose induction. To confirm the expression and examine the subcellular localisation of the transformed constructs, cells were viewed by confocal microscopy after 6 hours of induction of protein expression. (A) CG10806-YFP shows expression at both the plasma membrane and vacuolar membrane. (B) CG31052-YFP shows expression at the vacuolar membrane. (C) A western blot of the expressed proteins using an anti-GFP antibody. (D,E) Rescue of exchanger double-mutant yeast by Drosophila CPA2 genes was assessed by serial tenfold dilutions in 200 mM NaCl (D) or 1 M KCl (E) and comparison with wild-type (NHA1 NHX1) or exchanger double-mutant (nha1 nhx1) cells. The control NHX1 NHA1 strain G19 was transformed with empty pYes2.1 and the nha1 nhx1 mutant strain AXT3 was transformed with empty pYes2.1 or with YFP-tagged KHA ORFs. After induction of protein expression for 6 hours, cultures were adjusted to OD600=1 and further serial tenfold dilutions were made. Equal volumes were then spotted onto selective media containing either 200 mmol/l NaCl (D) or 1 mol/l KCl (E). Cultures were then allowed to grow for a further 3 days before imaging.

 

Figure 7
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  		Fig. 7. Overexpression of CG10806 but not CG31052 in tubule principal cells confers a fluid-secretion phenotype. Basal and neuropeptide-stimulated fluid-secretion rates were measured in c42>CG10806-RB::eYFP (A) or c42>CG31052::eYFP (B) Malpighian tubules and compared to the UAS parents. Basal secretion rates were assessed for 30 minutes before tubules were challenged with the diuretic peptide Capa-1 (100 nM). Secretion rates were then measured for a further 1 hour. (C) As in A, but tubules were stimulated with 1 µM cGMP at the time shown. n=9-12 for each experiment; error bars represent the s.e.m.

 

Figure 8
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  		Fig. 8. Overexpression of CG31052, but not CG10806, affects both Na+ and K+ handling in the Drosophila renal tubule. Transgenic tubules were dissected and secreted fluid collected over 1 hour under resting conditions. Na+ and K+ were measured by flame photometry, and are compared with the actinGAL4 parental control. Data are expressed as mean ± s.e.m. of four replicates. Where error bars are not shown, they are too small to be visible. Significant changes (P<0.05) relative to parental controls are marked with an asterisk.

 

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© The Company of Biologists Ltd 2008