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	Figure 1. A new family of sea urchin ADP-ribosyl cyclases.A, Sequence alignment of the predicted coding regions of three ADP-ribosyl cyclases isolated from Stronglycentrotus purpuratus cDNA libraries (termed SpARC1-3) and representative members of the ADP-ribosyl cyclase family. Conserved cysteine and catalytic residues are highlighted in yellow and green, respectively. Abbreviations: Ac, Aplysia californica; Hs, Homo sapiens; Sm, Schistosoma mansoni.
	Figure 2. SpARC1 is glycosylated.A, In vitro translation. mRNA coding for C terminally myc-tagged SpARC1 was translated in the presence of [35S]methionine using rabbit reticulocyte lysate. Autoradiogram of translation reactions performed in the absence (−) or presence (+) of canine pancreatic microsomes. B, Expression of SpARC1 in Xenopus laevis embryos. Western blot analysis using an anti-myc antibody of homogenates prepared from control embryos (C) or embryos injected with mRNA for myc-tagged SpARC1. Samples were either mock treated (−) or digested with PNGase F (+) prior to analysis (left). Migration of molecular mass markers (in kDa) is shown on the left of the panels.
	Figure 3. SpARC1 is a soluble luminal protein.A, Sub-cellular fractionation. Xenopus laevis embryos expressing myc-tagged SpARC1 were homogenized in a sucrose-containing buffer and diluted in to the same buffer (C), a carbonate-containing buffer (Na2CO3) or a hypertonic buffer (Hyper.). In other experiments the embryos were freeze-thawed and sonicated in a hypotonic buffer (Hypo.). All samples were centrifuged and the resulting pellets and supernatant fractions analysed by Western blotting. B, Proteolytic protection. Xenopus laevis embryos expressing myc-tagged SpARC1 were homogenized in a sucrose-containing buffer and the homogenates incubated with or without 1% v/v Triton X-100. Samples were then treated with the indicated concentration of proteinase K before Western blot analysis.
	Figure 4. SpARC1 is not secreted.Control Xenopus laevis oocytes or oocytes injected with myc-tagged SpARC1 were cultured overnight and Western blots for both the oocytes (O) and culture media (M) performed. 0.5 oocyte equivalents were loaded in each lane.
	Figure 5. SpARC1 localizes to the endoplasmic reticulum in Xenopus laevis oocytes and eggs.A, Confocal fluorescence images of Xenopus laevis oocytes co-expressing myc-tagged SpARC1 (red) and GFP-tagged human reduced folate carrier (hRFC, green). SpARC1 expression was detected by immunocytochemistry using an anti-myc primary antibody and an FITC labeled secondary antibody whereas hRFC was detected by GFP fluorescence. A plot of the individual emission intensities with depth averaged along a 5 µm wide section (arrowed line) in the overlay image is shown on the right. B, Lateral (‘xy’) images of SpARC1 expression (green) in oocytes and in eggs (right). The control image was obtained from an identically processed un-injected oocyte. Images were captured from the animal (A) and vegetal (V) poles as indicated.
	Figure 6. SpARC1 localizes to the endoplasmic reticulum in HEK cells.A, Western blot analysis using an anti-myc antibody of homogenates prepared from mock transfected cells (C) or cells transfected with myc-tagged SpARC1 (left). PNGase F treatment (right) was performed as described in Fig. 2. B–C, Expression of myc-tagged SpARC1 and DsRed2-ER (B) or mitochondrial GFP (C) in HEK cells transfected with the indicated plasmid or plasmid combination. SpARC1 expression was detected by immunocytochemistry using an anti-myc primary antibody and a Cy5-labelled secondary antibody, whereas the endoplasmic reticulum and mitochondria were visualized by fluorescence of the DsRed2 and GFP reporters, respectively. Results from control, mock-transfected cultures are also shown. Images were captured at the wavelengths corresponding to the colours marked at the side of the figure.
	Figure 7. SpARC1 catalyses NAADP production.A, Hypotonic lysates from Xenopus laevis embryos were incubated with NADP (1 mM) and nicotinic acid (NA, 50 mM) at pH 4 for 21 h and the resulting mixtures separated by HPLC. The top chromatograms are from lysates prepared from control embryos (dashed lines) and embryos injected with myc-tagged SpARC1 mRNA (solid line). The elution of authentic NAADP is shown below. B, Western blot comparing SpARC1 expression in 4 independent embryo preparations. C, Production of NAADP following incubation of a typical embryo homogenate with substrates for the indicated times. D, A plot of the initial rate of NAADP production (measured over 3 h) against the level of SpARC1 expression (determined by densitometric analysis of Western blot data for the preparations analysed in B).
	Figure 8. SpARC1 catalyses cyclic GDP-ribose production.Homogenates from Xenopus laevis embryos were incubated with 1 mM NGD for 21 h at pH 7.2 and the resulting mixtures separated by HPLC. A, Representative chromatograms from lysates prepared from control embryos (dashed lines) and embryos injected with myc-tagged SpARC1 mRNA (solid line). Both hypotonic lysates (Hypo.; top) or homogenates prepared in sucrose-containing medium (Sucrose; middle) were used. The elution of authentic cyclic GDP-ribose is shown below. B, Pooled data (mean+/−standard error of the mean) from 3 experiments comparing the amount of cyclic GDP-ribose produced using either control (C) or SpARC1-expressing preparations. C, Western blot analysis of myc-tagged SpARC1 expression in pellet (P) and supernatant (S) fractions prepared following centrifugation of the two reaction mixes from a typical experiment described in A at the end of the incubation period. D, 45Ca flux uptake by homogenates prepared from Xenopus embryos (in sucrose-containing media) or sea urchin eggs either in the absence (−) or presence (+) of the sarco-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin (Tg; 1 µM). Data are presented as means±standard deviation of triplicate samples from one of 3 independent experiments.
	Figure 9. Compartmentalized ADP-ribosyl cyclase signalling.Hypothetical scheme whereby putative transporter molecules allow delivery of substrates from the cytoplasm (Cyto.) to SpARC1 located in the endoplasmic reticulum (ER) and the subsequent release of the formed products. See text for details.

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