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Physiol. Genomics 26: 35-45, 2006. First published April 11, 2006; doi:10.1152/physiolgenomics.00038.2006
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Received 1 March 2006; accepted in final form 9 April 2006.
Physiological Genomics 26:35-45 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Novel subcellular locations and functions for secretory pathway Ca2+/Mn2+-ATPases

Tony D. Southall *, Selim Terhzaz *, Pablo Cabrero , Venkateswara R. Chintapalli , Jennifer M. Evans , Julian A. T. Dow and Shireen-Anne Davies

Division of Molecular Genetics, Anderson College Complex, University of Glasgow, Glasgow, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Secretory pathway Ca2+/Mn2+-ATPases (SPCAs) are important for maintenance of cellular Ca2+ and Mn2+ homeostasis, and, to date, all SPCAs have been found to localize to the Golgi apparatus. The single Drosophila SPCA gene (SPoCk) was identified by an in silico screen for novel Ca2+-ATPases. It encoded three SPoCk isoforms with novel, distinct subcellular specificities in the endoplasmic reticulum (ER) and peroxisomes in addition to the Golgi. Furthermore, expression of the peroxisome-associated SPoCk isoform was sexually dimorphic. Overexpression of organelle-specific SPoCk isoforms impacted on cytosolic Ca2+ handling in both cultured Drosophila cells and a transporting epithelium, the Drosophila Malpighian (renal) tubule. Specifically, the ER isoform impacted on inositol (1,4,5)-trisphosphate-mediated Ca2+ signaling and the Golgi isoform impacted on diuresis, whereas the peroxisome isoform colocalized with Ca2+ "spherites" and impacted on calcium storage and transport. Interfering RNA directed against the common exons of the three SPoCk isoforms resulted in aberrant Ca2+ signaling and abolished neuropeptide-stimulated diuresis by the tubule. SPoCk thus contributed to both of the contrasting requirements for Ca2+ in transporting epithelia: to transport or store Ca2+ in bulk without compromising its use as a signal.

intracellular calcium; confocal microscopy; Malpighian tubule


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CA2+ ACTS AS A DYNAMIC SIGNALING MOLECULE in eukaryotic cells to control a diverse array of cellular functions (7). Additionally, the luminal concentration of Ca2+ in organelles such as the endoplasmic reticulum (ER) and Golgi apparatus is important for proper transcription and correct processing of proteins through the secretory pathway (12). The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and plasma membrane Ca2+-ATPase (PMCA) pumps, which transport Ca2+ into the ER and out of the cell, respectively, have been well characterized (51, 57). The secretory pathway Ca2+/Mn2+-ATPase (SPCA) family forms a distinct third class of Ca2+ pumps (Fig. 1A) (5). In contrast to SERCA pumps, they have an equal selectivity for transporting Ca2+ and Mn2+, translocate only one Ca2+ (or Mn2+) per ATP molecule hydrolyzed, and are insensitive to potent SERCA inhibitors such as thapsigargin (43).


Figure 1
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  		Fig. 1. Identification of the SPoCk gene as a secretory pathway Ca2+/Mn2+-ATPase (SPCA) family member and the annotation and genomic context of SPoCk transcripts. A: sequence similarity analysis of Ca2+-ATPase protein sequences. The tree demonstrates the distinct clustering of the three main subtypes of Ca2+-ATPases. The clustering correlated with the intracellular localization of the proteins. The Drosophila gene SPoCk clearly fell within the SPCA group of Ca2+-ATPases. Sequences were aligned using ClustalW and displayed using TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). PID Accession Numbers, beginning with S. cerevisiae ATPase, Ca2+ transporting, type 2C, member 1 (PMR1), are given in a clockwise order: 6321271, 3138890, 7595749, 12644373, 285369, 40788333, 7296577, 21287896, 3875247, 7291680, 3878521, 3211977, 114305, 2826866, 114312, 1083756, 5714364, 14286104, 7304318, and 3549723. Although the SPoCk-A transcript (SPoCk-trA) was used for this alignment, the other transcripts (SPoCk-trB and SPoCk-trC; which are identical over their conserved areas) aligned similarly. SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; PMCA, plasma membrane Ca2+-ATPase. B: alignment of the first 25 amino acids residues of SPoCk-C with Drosophila ubiquitin and Parkin. Dark shading of residues indicates exact matches between all sequences; lighter shading indicates conserved substitutions. C: annotation and genomic context of SPoCk transcripts. The newly discovered (Fig. S2) alternatively spliced transcripts (transcripts D–F) did not encode proteins and were not represented in Drosophila expressed sequence tag databases. Shaded areas indicate coding regions. F1 and R1 show the areas to which the primers for transcripts A and C–F bind; F2 and R2 show the areas to which the primers for transcript B bind.

 
The archetype of the SPCA family, the ATPase, Ca2+ transporting, type 2C, member 1 gene (Pmr1 gene), was identified in the yeast Saccharomyces cerevisiae (41), and, since then, the majority of physiological studies of SPCAs have been in yeast. PMR1 has been localized to the Golgi apparatus (2) and is required to provide Ca2+ and Mn2+ for correct protein sorting and degradation in the secretory pathway (16). Interestingly, Pmr1 mutants also rescue superoxide dismutase mutants and inhibit reverse transcriptase via an increase in cytosolic Mn2+ (8, 27).

PMR1 homologs have since been discovered in a diverse range of organisms, including the rat (19), the nematode Caenorhabditis elegans (32), and human (21, 39), and all have been found to localize to the Golgi apparatus. In humans, there are two SPCA genes [ATPase, Ca2+ transporting, type 2C, member 1 (ATP2C1) and member 2 (ATP2C2)], of which mutations in ATP2C1 have been identified to cause the skin disorder Hailey-Hailey disease (21), emphasising the importance of Ca2+ in maintaining epithelial integrity. Recently, the human SPCA2 (ATP2C2) gene has been functionally characterized (32), and complementation studies in yeast have suggested its importance in cellular Mn2+ detoxification (53). Overexpression of the C. elegans SPCA in COS-1 cells has shown that SPCA can establish baseline cytosolic Ca2+ oscillations without the involvement of the ER (32). Furthermore, recent evidence has shown the functional importance of the Golgi apparatus as a distinct, agonist-sensitive, intracellular Ca2+ store independent of the ER (52). SPCAs are thus important proteins involved in Ca2+ and Mn2+ homeostasis, Ca2+ signaling, and possibly the regulation of Mn2+-mediated removal of superoxide radicals.

In the present study, we sought representatives of the Ca2+-ATPase family within the Drosophila genome. We identified and characterized the single Drosophila SPCA gene and showed that it encoded three isoforms. We documented, for the first time, novel subcellular locations for SPCAs in the ER and peroxisomes in addition to the Golgi apparatus. This unexpected finding implies a further level of complexity in the maintenance of ER Ca2+ levels and also uncovers the requirement for high levels of Ca2+ and/or Mn2+ within peroxisomes. Additionally, we investigated the role of transcript-specific SPoCk isoforms both in Drosophila S2 cells and in an organotypic context, the Drosophila Malpighian (renal) tubule. Modulation of expression levels, combined with functional analysis, showed that this single gene provides an alternative route to Ca2+ refilling of ER and allows peroxisomes to sequester very high levels of Ca2+ and that in specialized Ca2+-transporting epithelia like the Drosophila tubule, the Golgi may play a significant role in Ca2+ signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fly stocks, S2 cell culture, and transfections.
Drosophila melanogaster strains were maintained on standard cornmeal-yeast-agar medium at 25°C with a 12:12-h light-dark photoperiod and 55% relative humidity. Transgenic flies tubules from 7-day-old adults were dissected in Schneider's medium (Invitrogen; Paisley, UK). S2 cells (Drosophila Schneider cell line 2, an embryonic cell line of D. melanogaster) were cultivated in Schneider's Drosophila medium containing 10% fetal calf serum and were transiently transfected using a calcium phosphate transfection kit (Invitrogen).

Data mining.
A low-stringency BLAST search of the Berkeley Drosophila genome database (http://www.fruitfly.org) using the S. cerevisiae PMR1 and human ATP2C1 amino acid sequences identified the gene CG7651 (chromosome 3, cytological position 80A1–80A2). Complete amino acid sequences corresponding to other SPCA, SERCA, and PMCA sequences were obtained from the Swiss-Prot and National Center for Biotechnology Information sequence banks. Sequences were aligned using the program ClustalX, and a sequence similarity phylogenetic tree was generated using Tree View (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Generation of overexpression constructs for use in S2 cells and transgenic flies.
The open reading frame of SPoCk-A was PCR amplified from the expressed sequence tag (EST) LD03227 using a reverse primer containing the coding sequence for the c-myc epitope. This was TOPO cloned into the pMT/V5-His vector (Invitrogen) for expression in S2 cells. The open reading frames of SPoCk-B and SPoCk-C were PCR amplified from ESTs RH52668 and RE31249, respectively, fused to enhanced green fluorescent protein (GFP) and enhanced yellow fluorescent protein (YFP) using fusion PCR (creating a COOH-terminal fusion), and cloned into the pMT/V5-His TOPO vector. The three tagged transcript sequences were also cut out from the pMT/V5-His vector and cloned into the pUAST vector (46) to allow the generation of transgenic flies. Transgenic lines were generated under standard protocols (10, 29). For S2 cells, construction of the aequorin and Drosophila leucokinin receptor (dLKR) S2 expression plasmids was carried out as previously described (38, 40).

Peptide antibody production.
Rabbit anti-peptide antibodies were raised against the SPoCk-A epitope VDHDVLKQKPRNVK (residues 737–750), SPoCk-B epitope KQLKNQLEQKQILP (residues 98–111), and SPoCk-C epitope RTISIFIEEQTGLT (residues 6–19) by Genosphere Biotechnologies (Paris, France). Antisera were purified on a HiTrap NHS-activated HP column (Amersham Pharmacia Biotech; Buckinghamshire, UK) according to the manufacturer's instructions.

Immunofluorescence and immunoblot analysis.
The protocol used for immunohistochemistry and Western blot analysis was as described previously (10, 29, 38), and the following primary antibodies or dyes were used: affinity-purified rabbit anti-SPoCk-A, anti-SPoCk-B, and anti-SPoCk-C (1:1,000); rabbit anti-catalase (1:500, Abcam; Cambridge, UK); rabbit anti-COP II (1:250, Abcam); mouse anti-GFP (1:1,000, Zymed, now Invitrogen); mouse anti-c-myc (1:2,000, Cell Signaling; Danvers, MA); ER tracker dye (1 µM for 15 min, Molecular Probes, now Invitrogen); 4',6-diamidino-2-phenylindole (1 µg/ml for 1 min, Sigma; Poole, UK); and brefeldin A (BFA; 20 µM for 1 h, Sigma). Slides were viewed using a Zeiss 510 META confocal microscope.

Cellular fractionation.
Peroxisomal fractions was prepared according to standard techniques (18). Briefly, 1 g of whole flies overexpressing the SPoCk-C transgene was homogenized with a Polytron homogenizer in 0.25 M sucrose, 5 mM MOPS, and 1 mM EDTA (adjusted to pH 7.4 with NaOH) containing 0.1% (vol/vol) ethanol and 0.2 mM DTT plus protease inhibitors [0.2 mM PMSF and protease inhibitor cocktail (Sigma)]. The total homogenate was subjected to differential pelleting: nuclei, mitochondria, and large sheets of membrane were removed by two successive centrifugations at 1,950 g for 10 min and the crude peroxisomal fraction was pelleted by centrifugation at 25,000 g for 30 min. The crude peroxisomal fraction was carbonate treated to isolate the integral membrane proteins of peroxisomes.

Real-time cytosolic Ca2+ measurement.
Luminometry experiments were carried out on transfected S2 cells and live, intact tubules expressing aequorin (aeq) transgenes as previously described (25, 30, 36, 38, 40, 45). For tubule experiments, flies of the following genotypes were used (UAS, upstream activating sequence): 1) w–; UAS-aeq/+; +/+; c42/c42; 2) w–; UAS-aeq/+; SPoCk-A-c-myc/+; c42/+; 3) w–; UAS-aeq/+; SPoCk-B-GFP/+; c42/+; 4) w–; UAS-aeq/+; SPoCk-C-YFP/+; c42/+; and 5) w–; SPoCk-interfering RNA (RNAi)/+; c42/+.

Luminal Ca2+ flux measurement.
45Ca2+ (0.05 µCi, Amersham Pharmacia Biotech) was added to tubule secretion assays, which were performed as previously described (15). Secreted fluid was collected for 100 min; the volume was measured and counted in Optiflow SAFE scintillant (Fisher Scientific) for 1 min. Transport ratios were calculated as the ratio of the specific activity of a secreted drop to a reservoir bubble. Ratios >1 imply concentration of Ca2+ by the tubule, and, because the tubule lumen is positive relative to the blood side by typically 30–50 mV (34), thus prove transepithelial active transport.

Basolateral Ca2+ flux measurement.
Methods for Ca2+ storage measurements have been described in detail by Dube et al. (5). Briefly, eight pairs of Malpighian tubules were dissected in Schneider's medium (Invitrogen) and placed under liquid paraffin oil into 10-µl droplets of bathing medium containing 45Ca2+. After an exposure time of 60 min, tubules were briefly washed in "cold" Ca2+-free saline containing 2 mmol/l EGTA, radioactivity was counted in Optiflow SAFE scintillant (Fisher Scientific), and the 45Ca2+ content was determined.

Renal fluid secretion assays.
Fluid transport assays were carried out as previously described (13) using live, intact tubules dissected from 7-day-old adults with the following genotypes: 1) w–; +/+; c42/c42; 2) w–; SPoCk-A-c-myc/SPoCk-A-c-myc; +/+; 3) w–; SPoCk-A-c-myc/SPoCk-A-c-myc; c42/c42; 4) w–; SPoCk-RNAi/+; c42/+; and 5) w–; SPoCk-RNAi/SPoCk-RNAi; +/+. Fluid droplets were collected every 10 min, and the volumes of fluid were calculated. Basal rates of fluid secretion were monitored for 30 min, whereupon capa-1, an endogenous Drosophila neuropeptide that stimulates Ca2+ signaling and fluid transport (23), was added at 10–7 M, and the secretion rate was then recorded for a further 30 min.

Real-time RT-PCR and quantitative RT-PCR.
For real-time RT-PCR (Supplemental Fig. S1),1 primers 5'-GCCTTCCGCGCCCCCCTGCAAGCTGTC-3' and 5'-GGCCACCGTGACCACAATTAGGATGGC-3' were used to detect SPoCk-A and SPoCk-C, and primers 5'-CCCAAGACCTCGATCTGAGTTTC-3' and 5'-CAAGAAGCAGCAGAATTAGCGG-3' were used to detect SPoCk-B. Total RNA extraction and reverse transcription were performed as described previously (11). PCRs were performed with 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min.

For quantitative RT-PCR validation of over- or underexpression in transgenic Drosophila, primers 5'-AAGAAGGCGGACATCGGCATAG-3' and 5'-TGTTTGAGCACATCGTGGTCG-3' were used to detect and quantify SPoCk-A and SPoCk-C and primers 5'-TTTTCATTGAGGAACAGACGGG-3' and 5'-CACCAGAGCAGAACCAAGAAG-3' for SPoCk-B only. mRNA was prepared from 7-day-old Oregon R tubules using a Qiagen RNAeasy column following the manufacturer's methods (Qiagen; West Sussex, UK). Reverse transcription was carried out using Superscript II (Invitrogen) using oligo-dT primer. For each sample, 500 ng of cDNA were added to 12.5 µl of 2 µM SYBR green reaction mix (Finnzymes, GRI; Essex, UK) and 1 µl of a 6.6 µM concentration of the appropriate primers. An Opticon 2 thermocycler (MJ Research, now Bio-Rad; Herts, UK) was set as follows: 95°C for 15 min followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. The ribosomal protein 49 (rp49) gene was used as a standard in all experiments. For each condition, four to six independent samples were used, and each PCR was repeated three times independently to ensure the reproducibility of the results. For data analyses, the threshold cycle and sample quantity calculations were exported into a Microsoft Excel Worksheet for further statistical analysis.

Construction of the SPoCk-RNAi transgene.
To produce a construct for the transgenic RNA interference of SPoCK, we cloned an inverted repeat of a 685-bp fragment of SPoCk (+25–710 bp of exon 3, which is common to all transcripts; see Fig. 1) into the P-element vector pWIZ (28). The following primers were used to generate the SPoCk region containing AvrII restriction sites on both ends: 5'-GGGGCCTAGGATGCTGTTGTCCACCTCGG-3' and 5'-GGGGCCTAGGGAAGACCTCACCGAACTCAC-3'. The PCR product was then subcloned into the pWIZ vector using AvrII and NheI sites as a tail-tail inverted repeat flanking the white intron. The pWIZ-SPoCk construct was transformed into Drosophila by standard methods (11, 29), and several transgenic lines were generated, each with an independent insertion of pWIZ.

Statistical analysis.
Data are presented as means ± SE. Where appropriate, statistical significance of differences was assessed with Student's t-test (two-tailed) for unpaired samples, taking P < 0.05 as the critical level. Significant differences are marked with an asterisk in the figures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In silico identification and expression analysis of the Drosophila SPCA gene.
The Drosophila SPCA gene (CG7651/CG14449/CG32451) was identified by BLASTP searching using the S. cerevisiae PMR1 amino acid sequence (48% identity) and human ATP2C1 sequence (59% identity). Alignment of the CG7651 gene product with PMR1 and ATP2C1 revealed that all the domains characteristic of a SPCA (32) were conserved. A sequence similarity tree generated using other SPCA sequences, and with SERCA and PMCA sequences, clearly placed the CG7651 gene product in the SPCA cluster (Fig. 1A). There were no other Drosophila genes closer to the SPCA cluster than to other classes of ATPase. This sequence analysis showed that CG7651 does indeed encode the single Drosophila SPCA. In tradition with previous nomenclature of Drosophila genes, this gene was named SPoCk as it sits next to a gene called Jim.

Online EST data revealed three alternatively spliced transcripts for SPoCk, one of which contained an extra coding sequence that would result in proteins with an extended NH2-terminus (Fig. 1C). CG32451-PB (SPoCk-B) encoded an additional 133 amino acids, whereas CG32451-PC (SPoCk-C) encoded an extra 23 amino acids. The SPoCk-B NH2-terminal domain appeared to share no sequence similarity with any other protein, whereas the SPoCk-C NH2-terminal domain was similar to repeated regions in several Drosophila ubiquitin-like proteins, such as polyubiquitin and Parkin (Fig. 1B).

Validation of these EST predictions by direct RT-PCR (Fig. S1) confirmed the existence of three major transcripts but additionally identified three further minor transcripts, which were cloned and validated by sequencing (Fig. 1C).

SPoCk-A was ubiquitously expressed in the fly, throughout development and in all tissues tested, whereas SPoCk-B and SPoCk-C had more restricted expression patterns and were not expressed in Malpighian (renal) tubules and testes, respectively (Fig. S1). Furthermore, real-time RT-PCR analysis elicited a greater expression of SPoCk-C relative to SPoCk-A in the Malpighian tubule compared with the whole fly (Fig. S1). The Malpighian tubule localization is of particular interest, because Ca2+ signaling in this tissue is understood in some detail (14), and so it was selected for subsequent phenotypic analysis.

Subcellular localization of SPoCk isoforms.
The overall similarity of the three deduced peptides argued against an antibody-based strategy, so to assign cellular functions and to study intracellular localizations of the SPoCk isoforms, each was COOH-terminally tagged and expressed both in Drosophila S2 cells and in transgenic Drosophila. SPoCk-A was tagged with a c-myc epitope, SPoCk-B with enhanced GFP, and SPoCk-C with enhanced YFP. Transgenic flies were generated that could express these tagged isoforms under the control of the UAS/GAL4 binary system (9). Given the role of human SPCA in epithelial function, phenotypic analysis focused on the Drosophila Malpighian (renal) tubule, which must actively transport Ca2+ at very high rates (15) without compromising its use as a second messenger (14). Like pancreatic acini (35), the tubule is thus an excellent model for aspects of metazoan Ca2+ handling that cannot be addressed in yeast or mammalian cell lines.

Confocal microscopy revealed the intracellular localization of the three isoforms when they were expressed in S2 cells (Fig. 2). Furthermore, Fig. 3 shows the in vivo expression and localization of SPoCk isoforms in principal cells of the Malpighian tubule, achieved by crossing SPoCk transgenic lines with the principal cell-specific GAL4 driver line c42 (10, 29, 40). SPoCk-A localized to the Golgi apparatus in S2 cells (Fig. 2, A–C) and in vivo (Fig. 3, a–d), as confirmed by substantial colocalization with the Golgi-specific antibody COPII (17) in S2 cells (Fig. 2C) and a polyclonal SPoCk-A antibody in principal cells of the tubule (Fig. 3, c and d). SPoCk-B localized to the ER (reticular network throughout the cytoplasm) in both S2 cells (Fig. 2, D–F) and tubule principal cells (Fig. 3, e–h). The ER localization was confirmed by colocalization with ER tracker dye (Invitrogen) in S2 cells (Fig. 2F) and in tubule principal cells (Fig. 3h). Although SPoCk-A and SPoCk-B were both found to ramify throughout the cell both in culture and in vivo, the two staining patterns were easily distinguished: the ER staining of SPoCk-B was markedly reticular (for example, in Fig. 3, e–h) compared with the more lobular structure for the secretory pathway isoform A (for example, in Fig. 3, a–d). By contrast, SPoCk-C was found to reside on large vesicular-like structures in both S2 cells (Fig. 2, G–I) and tubule principal cells (Fig. 3, i–l). The possibility that these might be lysosomes or pigment granules was initially considered. However, their large size, low abundance, and the presence of high levels of SPoCk-C transcript in the tubule (Fig. 3, i and j) suggested that these organelles could be peroxisomes. This was tested and confirmed by colocalization with catalase using an anti-catalase antibody (Fig. 3, j–l). Catalase, which is the most abundant peroxisomal protein, resides in the electron-dense peripheral matrix of peroxisomes (22), and the colocalization observed correlated well with the expected membrane localization of SPoCk-C. Additionally, the pattern of SPoCk-C fluorescence matched the description and abundance of peroxisomes in the tubule as reported by Beard and Holtzman (6). Use of a polyclonal antibody generated for the NH2-terminal region of SPoCk-C in wild-type tubules showed a similar staining pattern to those seen in Fig. 3, j and k, confirming its peroxisomal localization (Fig. 3i). Colocalization experiments using this antibody and catalase (data not shown) confirmed the peroxisomal localization.


Figure 2
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  		Fig. 2. Intracellular localization of SPoCk isoforms in S2 cells. A–C: Golgi apparatus localization of SPoCk-A. Shown is the colocalization (C) of SPoCk-A (c-myc tagged, green; A) with the Golgi-specific COPII antibody (red, B). D–F: endoplasmic reticulum (ER) localization of SPoCk-B. Shown is the colocalization (F) of SPoCk-B [green fluorescent protein (GFP) tagged, green; D] with blue ER tracker dye (Molecular Probes, blue; E). G–I: peroxisomal localization of SPoCk-C. Shown is colocalization (I) of SPoCk-C [yellow fluorescent protein (YFP) tagged, green; G] with catalase (red; H). Scale bars = 10 µm.

 

Figure 3
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  		Fig. 3. Intracellular localization of SPoCk isoforms in vivo within principal cells of the Malpighian tubule. a: Golgi apparatus localization of SPoCk-A. Shown is SPoCk-A in principal cells of the tubule (c-myc tagged, Texas red secondary antibody, red). b–d: colocalization (d) of SPoCk-A (c-myc tagged, FITC secondary antibody, green; b) with affinity-purified rabbit anti-SPoCk-A (red; c). e: ER localization of SPoCk-B. The nucleus is labeled blue with 4',6-diamidino-2-phenylindole (DAPI). f–h: Colocalization (h) of SPoCk-B (GFP tagged, green; f) with blue ER tracker dye (blue; g). i: Peroxisomal localization of SPoCk-C. Endogenous SPoCk-C in the tubule was detected with a SPoCk-C-specific antibody; nuclei are labeled blue with DAPI. j–l: Colocalization (l) of SPoCk-C (YFP tagged, green; j) with catalase (red; k). All scale bars = 10 µm. The specificity of anti-catalase and anti-COPII antibodies was verified by Western blot analysis (Fig. S2, D and E).

 
Expression of SPoCk isoforms was confirmed by Western blot analysis of transgenic fly lines overexpressing the tagged transgenes (Supplemental Fig. S2). The SPoCk-A antibody recognized a protein of the expected size (100 kDa). The SPoCk-B antibodies recognized both a protein of the expected size (140 kDa) and a stronger band at ~125 kDa. It is possible that the extra residues of SPoCk-B are cleaved off after a processing event. SPoCk-C protein (130 kDa) was only detected in a peroxisomal membrane protein preparation from SPoCk-C overexpressing flies. This provides further confirmation for the novel peroxisomal location of SPoCk-C.

Overexpression of different SPoCk isoforms modulates Ca2+ signaling in vivo and in culture.
The functional significance of a gene can usefully be explored by modulating its expression (viz reverse genetics). Both the ER and Golgi have been implicated as mobilizable Ca2+ stores, so the effect of overexpressing individual SPoCk isoforms on Ca2+ signaling in vivo was thus studied. In particular, we were interested in the mechanism of Ca2+ signaling in nonexcitable polarized epithelial cells, which is traditionally explained by the activation of inositol (1,4,5)-trisphosphate (IP3) signaling cascades and ER Ca2+ (42).

The UAS-aequorin system allows real-time quantitative measurement of Ca2+ signaling events both in Drosophila S2 cells (38) and in the Malpighian tubule (25, 40). To investigate the effects of SPoCk overexpression in S2 cells, cells were cotransfected with the bioluminescent calcium reporter UAS-aequorin, dLKR (38), and a tagged SPoCk isoform. Stimulation of S2 cells expressing dLKR, with the neuropeptide Drosophila leucokinin, elicited an increase in cytoplasmic Ca2+ via the IP3 pathway (36, 38). The Drosophila leucokinin response in S2 cells was unaffected by overexpression of SPoCk-A or SPoCk-C; however, overexpression of SPoCk-B significantly increased the intracellular Ca2+ concentration ([Ca2+]i) response from 271 ± 7 (control) to 371 ± 4 nM above basal levels (Fig. 4, A and B). This result shows that Ca2+ signaling in S2 cells is sensitive to levels of the SPoCk-B transcript, which must thus physiologically limit the normal response; if it played a minor role, then an increase of expression would not be expected to have a detectable impact on the Ca2+ signal. In S2 cells, the ER is believed to be the principal IP3-releasable store, whereas the Golgi does not play an important role in Ca2+ release (55). Thus it is likely that only the ER-localized SPoCk isoform, SpoCk-B, influenced cytosolic Ca2+ levels in S2 cells, as confirmed in Fig. 4.


Figure 4
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  		Fig. 4. Overexpression of transcript-specific SPoCk isoforms. A and B: S2 cells. S2 cells transfected with expression constructs for UAS-aequorin, the Drosophila leucokinin receptor, and a SPoCk isoform were challenged with Drosophila leucokinin at a concentration of 10–7 M (38). A: maximum intracellular Ca2+ concentration ([Ca2+]i) increase above resting levels in S2 cells expressing SPoCk-A, SPoCk-B, and SPoCk-C (means ± SE; N = 10). The Ca2+ increase in cells expressing SPoCk-B was significantly greater than in control cells (***P < 0.0001). B: for clarity, typical [Ca2+]i responses in control (38) and SPoCk-B-expressing cells are shown. C–F: in vivo principal cells of the Malpighian tubule. Shown is the targeted expression of SPoCk isoforms in transgenic tubules. C: typical biphasic capa-1 [10–7 M (23)] response in tubule principal cells overexpressing SPoCk-A (red, aeq/+; SPoCk-A/+; c42) compared with a typical control response (black, aeq; +; c42). D: typical biphasic capa-1 response in tubule principal cells overexpressing SPoCk-B (red, aeq/+; SPoCk-B/+; c42/+) compared with a typical control response (black, aeq; +; c42). E: typical biphasic capa-1 response in tubule principal cells overexpressing SPoCk-C (red, aeq/+; SPoCk-C/+; c42) compared with a typical control response (black, aeq; +; c42). F: bar graph showing the maximum increase of [Ca2+]i above resting levels in principal cells for both the primary and secondary responses (means ± SE; N ≥ 11). Overexpression of SPoCk-A resulted in a significantly increased primary response (**P = 0.0043). Overexpression of SPoCk-A (**P = 0.0011), SPoCk-B (**P = 0.0039), and SPoCk-C (***P < 0.0001) resulted in a significantly decreased secondary response. G–I. effect of brefeldin A (BFA) on Golgi complex structure and the capa-1-induced [Ca2+]i response. Disruption of the Golgi complex structure by pretreatment for 1 h with 20 µM BFA was shown by the reduced staining of the c-myc epitope in BFA-pretreated SPoCk-A-overexpressing tubules (H) compared with control tubules (G). Scale bars = 10 µm. I: typical capa-1 (applied at a concentration of 10–7 M) response in principal cells overexpressing SPoCk-A compared with a typical control (c42UAS-aeq) response. The application of BFA (20 µM) resulted in a significantly lower increase in both SPoCk-A-overexpressing (***P = 0.0006) and wild-type tubules (***P = 0.0006) compared with untreated tubules.

 
The Malpighian (renal) tubule provides a robust phenotype for the integrative physiology of cell signaling and transport genes (14, 25), which allows investigations of Ca2+ signaling in an organotypic context using targeted Ca2+ reporters. Previous work using targeted UAS-aequorin has revealed distinct cell-specific intracellular Ca2+ signatures in adjacent cells in the same tubule (40). Furthermore, analysis of neurohormone control of the tubule has revealed modulation of cell-specific Ca2+ signaling by different endogenous neuropeptides (23, 40, 45). The Drosophila neurohormone capa-1 acts upon the principal cells of tubules (23) via an IP3-mediated pathway (36). However, although the involvement of IP3 in tubule Ca2+ signaling and fluid transport is not in doubt, the precise intracellular Ca2+ pools that contribute to tubule function have not been determined.

Transgenic fly lines containing both UAS-UAS-aequorin and UAS-SPoCk transgenes were crossed to GAL4 enhancer trap insertion c42 to drive expression in tubule principal cells (25, 36, 40). Cytoplasmic Ca2+ was then monitored in live, intact tubules from the resulting offspring (40) to provide organotypic Ca2+ measurements.

Data in shown Fig. 4, C–E (summarized in F) show the impact of SPoCk isoform-specific overexpression on the capa-1-induced [Ca2+]i response in tubules. In contrast to S2 cells, overexpression of SPoCk-B did not increase the IP3-induced primary peak; however, the overexpression of SPoCk-A significantly increased Ca2+ levels from 152 ± 11 (control) to 218 ± 16 nM above basal levels. Again, this increase in response implies that SPoCk-A, and by implication Golgi/secretory pathway stores, plays a significant role in Ca2+ signaling by the principal cell.

BFA is an antibiotic drug that inhibits the formation of the Golgi apparatus (31) and so has the potential to confirm the role of the Golgi network in fluid secretion by intact Malpighian tubule. Figure 4, G and H, shows the impact of BFA pretreatment of SPoCk-A overexpressing tubules compared with the control tubules. Staining of the c-myc epitope was reduced by BFA treatment, reflecting the fragmentation and subsequent disappearance of the Golgi apparatus in treated tubules (Fig. 4H). This disruption of the Golgi apparatus elicited a significant decrease in capa-1-induced Ca2+ release in control tubules. This effect was potentiated in tubules overexpressing the Golgi-specific SPoCk-A isoform (Fig. 4I), confirming that overexpressed SPoCk-A was localized to the Golgi.

The exclusive localization of SPoCk-A in the Golgi implies that in this renal tubule, it is not the ER, but the Golgi apparatus, that acts as the primary IP3-releasable pool. It also suggests that SPoCk-A plays an important role in maintaining a functional Ca2+ signaling pool in this renal epithelium.

Figure 4E shows that overexpression of all three SPoCk isoforms resulted in a reduction in the magnitude of the slow secondary Ca2+ rise. This may be due to an increased buffering of [Ca2+]i in the cell produced by the extra Ca2+-ATPase activity.

Functional correlates of SPoCk activity.
Because diuresis is known to be controlled by Ca2+, an increase in principal cell Ca2+ should be translated into enhanced fluid production. Figure 5A shows that overexpression of SPoCk-A significantly increased basal rates of fluid transport by tubule as well as the response to neuropeptide stimulation. Taken together with the previous results, we propose that the Golgi complex in principal cells contributes significantly to normal neuropeptide-induced Ca2+ signaling in the insect renal tubule.


Figure 5
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  		Fig. 5. Functional correlates of SPoCk activity. A: fluid transport by the Drosophila renal tubule is potentiated by overexpression of the Golgi-specific SpoCk-A transcript. Tubules in which UAS-SPoCk-A is driven by c42 (red) are compared with the UAS-SPoCk-A (blue) and c42 (black) parental lines. Tubules were stimulated at 30 min by the addition of 10–7 M CAPA neuropeptide. Data are shown as means ± SE; N = 10. B–E: peroxisome-specific SPoCk-C isoform is associated with Ca2+ storage excretion. B: confocal image of the initial segment of the anterior tubule. C: bright-field image. The lumen of the tubule is packed with SPoCk-C-positive concretion bodies. Nuclei are stained blue with DAPI. D: high-power view of the tubule initial segment showing the annular nature of anti-SPoCk-C staining. E and F: sexually dimorphic SPoCk-C expression in tubules. E: mRNA expression of SPoCk-C in female anterior tubules was significantly higher than in males and was increased compared with female posterior (***P < 0.0001), male anterior (**P = 0.0015), and male posterior tubules (**P = 0.001). Data are shown as means ± SE; N = 4. F: SPoCk-C protein expression was significantly increased in female anterior tubules by ~32% [band intensity determined using ImageJ sotftware (http://rsb.info.nih.gov/ij/)] compared with male anterior tubules. G and H: Ca2+ storage (G) and transport (H) of 45Ca2+ in SPoCk-C-overexpressing tubules. Tubules were incubated in radioactively labeled Ca2+, and the "normalized storage ratio" was calculated as the ratio of specific activities of the tubules to bathing fluid. Overexpression of SPoCk-C resulted in a significantly increased Ca2+ storage compared with the parental UAS-SPoCk-C (*P < 0.014) and c42 (*P < 0.033) parental lines. Data are shown as means ± SE; n ≥ 4 tubules. The "transport ratio" was calculated as the ratio of specific activities of the secreted fluid to that of bathing fluid. Overexpression of SPoCk-C resulted in a significantly increased Ca2+ transport compared with parental UAS-SPoCk-C (*P < 0.014) and c42 GAL4 (*P < 0.04) lines. Data are shown as means ± SE; n = 8 tubules.

 
Expression of SPoCk-C correlated with another important property of the tubule: bulk Ca2+ transport and sequestration. Drosophila contain a pair of anterior tubules and a pair of posterior tubules in the abdomen, connected to the hindgut but free floating in the hemocoel. All regions of the tubules are capable of Ca2+ secretion, but the initial and transitional segments of the anterior tubules are capable of transporting the entire animal's Ca2+ content in <2 h (15). Although some Ca2+ is secreted in soluble form, most appears to be sequestered into vesicles, called spherites (50); these are commonly observed in insect epithelia (48). Antibodies to SPoCk-C labeled the periphery of such vesicles in the lumen of the initial segment of the tubule (Fig. 5, B–D). It is thus likely that spherites are formed as concretions within specialized peroxisomes with high levels of SPoCk-C in their membranes, so facilitating Ca2+ uptake and storage, and are then discharged into the tubule lumen.

Anterior tubules store higher levels of Ca2+ than posterior tubules (15). The respective levels of SPoCk-C transcript were consistent with this observation (Fig. 5E). However, we also found sexually dimorphic expression of SPoCk-C transcript (Fig. 5E) and protein (Fig. 5F) in anterior tubules, with expression in females being significantly higher than in males.

To assess the role of the SPoCk-C isoform in sequestering Ca2+ within the tubule and/or secreting Ca2+ in soluble form into the secreted fluid, tubules overexpressing SPoCk-C were incubated in 45Ca2+, and both storage in and Ca2+ excretion by isolated female anterior Malpighian tubules were measured. Ca2+-specific activities in female anterior Malpighian tubules were increased by ~40% in tubules overexpressing SPoCk-C in principal cells than the control parental tubules (Fig. 5G), demonstrating a role of SpoCk-C in sequestering Ca2+. Active transepithelial transport of Ca2+ was also increased by overexpression of SPoCk-C in principal cells (Fig. 5H). SPoCk-C thus participates in a pathway for sequestering and ultimately excreting Ca2+.

Hypomorphic or null alleles are also valuable in a reverse genetic approach, so to further investigate the in vivo roles of SPoCk, heritable RNAi technology was used to reduce SPoCk expression. Thus transgenic flies were generated using an inverted repeat construct under control of the UAS promoter in the germ line transformation vector pWIZ (28). Crossing of the UAS-SPoCk-RNAi line to the c42 GAL4 enhancer trap line (the specific principal cell driver) decreased the expression of SPoCk mRNA in tubules of the progeny. Validation of the SPoCk-RNAi line by quantitative RT-PCR analysis confirmed an ~70% decrease in SPoCk mRNA levels in c42/UAS-SPoCk-RNAi Malpighian tubules compared with the c42 and UAS-SPoCk-RNAi parental lines (Fig. 6A). This reduction in SPoCk mRNA resulted in an alteration of the capa-1-induced [Ca2+]i signature (Fig. 6, B and C). The magnitude of the secondary peak was increased, which, notably, is the opposite phenotype from that resulting from SPoCk overexpression (cf. Fig. 4F).


Figure 6
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  		Fig. 6. Effects of downregulation of SPoCk expression. A: expression of SPoCk was significantly reduced in c42/UAS-SPoCk-interfering RNA (RNAi) compared with the parental UAS-SPoCk-RNAi (**P = 0.0021) and c42 GAL4 enhancer trap line (*P = 0.015) parental lines. B: typical biphasic capa-1-induced (10–7 M) Ca2+ response in principal cells of SPoCk-RNAi tubules (red, aeq/+; SPoCk-RNAi/+; c42) compared with a typical control response (black, aeq; +; c42). C: bar graph showing the maximum increase of [Ca2+]i in principal cells for both the primary and secondary responses (means ± SE; N ≥ 10). The reduction in SPoCk expression did not affect the primary response but resulted in a significantly increased secondary response (*P = 0.015). D: reduction in SPoCk expression inhibited basal and capa-1-stimulated fluid transport rates in the Drosophila renal tubule.

 
Further phenotypic analysis of c42/UAS-SPoCk-RNAi Malpighian tubules revealed that these transported fluid at significantly reduced rates compared with either the c42 or UAS-SPoCk-RNAi parental lines, with a significantly reduced response to capa-1 (Fig. 6D). Again, this effect of RNAi-mediated knockdown is the opposite of that observed by overexpression (cf. Fig. 5A).

These data thus suggest that SPoCk-A is a significant player in the normal neuroendocrine response of this tissue.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Ca2+ pumps in the ER.
SERCA pumps are absent from a large number of eukaryotes, including fungi, protozoans, and plants. In these organisms, the ER is maintained by ATPases distinct from the PMCA, SERCA, and SPCA groups (for a review, see Ref. 51) and are possibly more related to more primitive Ca2+-ATPases. However, in insect and vertebrate organisms, it is well documented that the ER Ca2+ concentration is maintained by the SERCA pump. Therefore, the ER localization of SPoCk-B was surprising because there is an existing mechanism for pumping Ca2+ into the ER in Drosophila (the SERCA pump, encoded by the Ca-P60A gene). Nevertheless, the discovery of a PMR1-like ATPase in the ER is not wholly unprecedented; overexpression studies of calreticulin in HEK-293 cells have implied the additional presence of a thapsigargin-insensitive Ca2+-ATPase in the ER (3). Indeed, Arnaudeau and colleagues (3) suggested that this might be a member of the PMR1 family of Ca2+-ATPases. Our discovery of the ER-targeted SPCA of Drosophila SPoCk-B sheds light on this mystery; it is likely that HEK-293 cells (and other cells in insects and vertebrates) possess an ER-targeted thapsigargin-insensitive Ca2+-ATPase that is either the product of an alternatively spliced transcript from a SPCA gene (like SPoCk-B) or is the product of a second SPCA gene [e.g., the ATP2C2/human SPCA2 gene in humans (47)]. The reason for the presence of another Ca2+-ATPase on the ER is unclear; it may be a part of a complex system for control of ER Ca2+ concentration or its primary role could be to maintain high levels of Mn2+ in the ER lumen.

Peroxisomes: vehicles for Ca2+ storage?
Peroxisomes are dynamic, self-replicating organelles enriched in tissues such as the kidneys and liver (for a review, see Ref. 37). Their well-conserved functions include H2O2-based respiration and fatty acid ß-oxidation. Over the past decade, a great deal has been learned about these enigmatic organelles, especially concerning the proteins involved in peroxisomal biogenesis and peroxisome biogenesis disorders (4). In Drosophila, they are implicated in the processing of visual pigments (44), a process that occurs in both the eye and Malpighian tubules (54). However, there is a lot that is not yet understood about the workings and functions of peroxisomes. The discovery of a peroxisomal Ca2+/Mn2+-ATPase in Drosophila (SPoCk-C) was unprecedented, and, presently, the role of this pump can only be speculated upon. One possibility is that SPoCk-trC is present to maintain high levels of Ca2+ and/or Mn2+ in peroxisomes and could be explained by the use of Ca2+ for the correct function of peroxisomal resident enzymes, such as Mn2+-dependent superoxide dismutase.

Peroxisomes as organelles for sexually dimorphic Ca2+ storage.
Another role of SPoCK-C for peroxisomes could be to maintain sufficient levels of Ca2+ to precipitate oxalates or phosphates, to form the Ca2+ spherites that are found in the initial segment of the anterior tubules. This is an example of "storage excretion." Normally, peroxisomes are involved in preventing the excess synthesis of oxalate, because a peroxisomal protein called alanine:glyoxylate aminotransferase (AGT) limits oxalate levels. Drosophila has such a peroxisomal enzyme, Drosophila serine:pyruvate aminotransferase (Dm-Spat), which functions as an AGT and may play a role in preventing the accumulation of glyoxylate (20). This gene is enriched by five times in the tubule relative to the whole fly, implying its importance in renal function (49).

Why is SPoCk-C more abundant in female Malpighian tubules? This probably reflects the extra demands of oogenesis for Ca2+, as the chorion is Ca2+ rich (35%) (24). The storage excretion of Ca2+ in spherites throughout life would thus provide an osmotically inactive bulk pool of Ca2+ that could be mobilized in adulthood, thus explaining the greater abundance of SPoCk-C vesicles in females.

It is known that the peroxisome originates from the ER by means of preperoxisomal vesicles (33) and that the ER is one of the intracellular Ca2+ pools. Thus the Ca2+ concentration in peroxisomes could be as high as in the ER, and the presence of the peroxisomal SPoCk-C isoform may be important in this regard. In this study, we have shown the important role of the overexpression of the SPoCk-C isoform in sequestering Ca2+ within the tubule and transporting it into the secreted fluid. Measuring the peroxisomal Ca2+ concentration and monitoring any link between the change of Ca2+ concentration in the cytoplasm and peroxisome should help in our understanding concerning Ca2+ storage in Malpighian tubules.

How is SPoCk-C targeted? Many peroxisomal proteins contain a peroxisomal targeting sequence; however, SPoCk-C does not contain any of these motifs. The targeting of SPoCk-C may be explained by the similarity of its extra 23 NH2-terminal amino acid residues to repeated domains of ubiquitin (Fig. 1B). It has been shown that the peroxisomal protein Pex4p is a ubiquitin-conjugating enzyme that is anchored to the cytoplasmic surface of peroxisomes (26) and can couple to ubiquitin in vitro (1). Pex4p is important for import of proteins into peroxisomes, and it is conceivable that the Drosophila homolog of Pex4p is involved in targeting of SPoCk-C by an interaction with this short ubiquitin-like NH2-terminal region.

The Golgi as a Ca2+ signaling organelle.
The Drosophila Malpighian tubule provides a powerful, tractable model for studying renal epithelium function. Transgenic manipulation of SPoCk-A levels has uncovered the important role of the Golgi in Ca2+ signaling events in a renal epithelium. The Golgi has been suggested to be an important Ca2+ store in renal LLC-PK1 cells (56); therefore, it appears that this may be a conserved feature of renal epithelia. We speculate that the relative importance of the Golgi in the tubule is because Ca2+ handling is problematic in a tissue that also transports and stores large amounts of Ca2+; perhaps the ER (and the derived peroxisomes) are freed to participate in transport roles by delegating the signaling function to the Golgi?

In conclusion, we identified, for the first time, localization of Ca2+/Mn2+-ATPases to the ER and peroxisomes as well as the Golgi. A single gene thus contributes to Ca2+ handling at three distinct intracellular sites; it provides an alternative route to Ca2+ refilling of the ER; it allows peroxisomes to sequester high levels of Ca2+ and subsequently transport it into the urine; and the data suggest that in specialized Ca2+-transporting epithelia like the Drosophila tubule, the Golgi apparatus plays a significant role in Ca2+ signaling.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by the United Kingdom Biotechnology and Biological Sciences Research Council in the form of grants (to S.-A. Davies and J. A. T. Dow) and studentships (to T. D. Southall and J. M. Evans).


   ACKNOWLEDGMENTS
 
Present address of T. D. Southall: Wellcome Trust/Cancer Research United Kingdom Gurdon Institute of Cancer and Developmental Biology, Tennis Court Rd., Cambridge CB2 1QN, UK.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S.-A. Davies, Div. of Molecular Genetics, Anderson College Complex, Univ. of Glasgow, Glasgow G11 6NU, UK (e-mail: s.a.davies{at}bio.gla.ac.uk).

* T. D. Southall and S. Terhzaz contributed equally to this work. Back

1 Supplemental material for this article is available at the Physiological Genomics web site. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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