Sequence alignment of lipid phosphate phosphatase proteins. The conserved phosphatase domains are shown in light blue, with those residues thought to be required for catalytic activity shown in dark blue (Brindley & Waggoner, 1998). The position of the WunD:248>T mutation is shown in white. Six transmembrane domains are indicated, as predicted by TMPred (http://www.ch.embnet.org/software/TMPRED_form.html; Hofmann & Stoffel, 1993), and are shown in boxes. Amino acids that are identical between all five sequences are indicated by asterisks, and are shown on a dark pink background or by dark pink letters. Where conserved substitutions have been identified, the amino acids are indicated by a colon, and are shown on a purple background or by purple letters; where semi-conserved substitutions have been identified, amino acids are indicated by a dot, and are shown on a green background or by green letters. The GenBank accession numbers for each sequence are as follows: human lipid phosphate phosphatase 1 (LPP1), (Kai et al., 1997); human LPP2, (Roberts et al., 1998); human LPP3, (Kai et al., 1997); Wunen, (Zhang et al., 1997); Wunen2, (Starz-Gaiano et al., 2001).

The Wunen protein and confirmation of protein sizes. (A) The Wunen (Wun) protein, indicating the position of the D:248>T point mutation. Conserved residues required for catalysis are shown in red (Neuwald, 1997). (B) Western blot confirming that each protein runs to the correct predicted size. GFP, green fluorescent protein; LPP, lipid phosphate phosphatase.

Ectopic expression in the mesoderm. Embryos were immunostained with anti-Vasa antibody to visualize the primordial germ cells (PGCs; brown) and with anti-green-fluorescent-protein (GFP) to visualize protein expression (blue). Embryos in (A–E) are viewed laterally, with the posterior pole to the right; embryos in (F–J) are viewed dorsally. Expression of Wunen (Wun)–GFP (A) or human lipid phosphate phosphatase 3 (LPP3)–GFP (B) at stage 10 results in an early loss of PGCs compared with the ectopic expression of WunD:248>T–GFP (C) or mouse Lpp1–GFP (D). These can be compared with a wild-type embryo (E) with a full complement of PGCs at the same stage. By the end of embryogenesis, those embryos expressing Wun–GFP (F) or LPP3–GFP (G) show a marked loss of PGCs. Embryos expressing WunD:248>T–GFP (H) or mouse Lpp1–GFP (I), however, show no apparent perturbation or loss of PGCs, and clearly form two gonads, as seen in a wild-type embryo at the same stage (J). Wild-type embryos have not been stained with anti-GFP, and consequently show no blue staining.

Phylogenetic tree of Wunen and Wunen 2 with the human lipid phosphate phosphatase isoforms. The tree was generated using the sequences shown in Fig. 1. LPP, lipid phosphate phosphatase.

PiPer® phosphate-release assay with lysophosphatidic acid. Fluorescence was measured using a SPECTRAmax™ GEMINI XS Dual Scanning Microplate Spectrafluorometer. (A) Mean fluorescent readout ± s.d. over time from the three experiments for each protein. The sequential action of the enzymes involved in the detection system after exposure to phosphate in solution accounts for the 20-min lag period seen at the start of the assay. Running phosphate standards alone produces the same lag period (see supplementary information online). Plotting phosphate standards against the corresponding fluorescent readout at the chosen timepoint (t = 65 min) gives a straight line in the presence of up to 4 nmol, indicating that the detection system shows first-order kinetics at this timepoint (B). These values are the means ± s.d. of the three samples for each standard. LPA, lysophosphatidic acid; LPP, lipid phosphate phosphatase; RFU, relative fluorescence units; Untrans, untransfected; Wun, Wunen.

Confirmation of proteins present at the cell surface. (A) Images obtained by confocal microscopy, showing Wunen (Wun)–green-fluorescent-protein (GFP), mouse lipid phosphate phosphatase 1 (Lpp1)–GFP and human LPP3–GFP at the surface of mesodermal cells in Drosophila embryos. Protein expression is driven by Twist–Gal4. (B) Biotinylation of human LPP3–GFP and mouse Lpp1–GFP at the surface of S2 cells, detected with anti-GFP antibody. E, elutions (protein that is biotinylated and has bound to the column); TL, total lysate before capture on the column; W, washes (protein that is not biotinylated and has not bound to the column).