 |
4, 8, 793–799 (2003)
doi:10.1038/sj.embor.embor900
Figures and Tables
Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo
Camilla Burnett & Ken Howard
| |
| | |
| |
|
| | |
| | Figures | |
| |
|
|
Figure 1
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), AB000888 (Kai et al.,
1997); human LPP2, AF047760
(Roberts et al., 1998); human LPP3,
AB000889 (Kai et
al., 1997); Wunen, U73822
(Zhang et al., 1997); Wunen2,
AF331162 (Starz-Gaiano
et al., 2001).
|
|
Figure 2
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.
|
|
|
Figure 3
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.
|
|
Figure 4
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.
|
|
|
Figure 5
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.
|
|
Figure 6
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).
|
|
|
| | |
| |
|
| | |
| | Tables | |
| |
| |
| | |
|
