Fig. 1. Purification of GPI-GnT complexes. GST–FLAG-PIG-A and FLAG-HAT-PIG-A were isolated by two-step affinity purification from the digitonin lysates of 1.4 × 10⁸ cells of JY5 transfectants. Samples of the purified proteins (equivalent to 6 × 10⁷ cells) were used for in vitro GPI-GnT assay (A) and the rest (equivalent to 8 × 10⁷ cells) were used for SDS–PAGE and silver staining (B). (A) Lysates derived from 10⁷ cells of wild-type JY25 as a positive control (lane 1) and purified complexes containing GST–FLAG-PIG-A (lane 2) or FLAG-HAT-PIG-A (lane 3) were incubated with radiolabeled UDP-GlcNAc and bovine PI. Lipids were analyzed by TLC. Identities of spots are indicated on the right. (B) Silver-stained SDS–PAGE profiles of purified complexes containing GST–FLAG-PIG-A (lane 1) and FLAG-HAT-PIG-A (lane 2). Band numbers are shown on the right. The positions of the molecular size markers are indicated on the left (kDa).

Fig. 2. (A) Amino acid sequence of human PIG-P. The sequence determined by protein sequencing is underlined. The DDBJ/EMBL/GenBank accession No. of human PIG-P cDNA is AB039659. (B) Hydropathy profile of human PIG-P (Kyte and Doolittle, 1982). (C) Alignment of amino acid sequences of human PIG-P and its homologs of A.thaliana, S.pombe and S.cerevisiae. Black and gray boxes indicate identical and similar amino acids, respectively. Amino acid numbers are indicated on the left. Two putative transmembrane regions of human PIG-P, TM1 and TM2 are indicated.

Fig. 3. (A) FACS analysis of 2.10 GPI (+), 2.10 GPI (–) and 2.10 GPI (–) cells transfected with pME-neo-PIG-P or pME-neo. Cells were stained by anti-Thy-1 (thick lines in a–d), isotype-matched control (thin lines in a–d), anti-CD48 (e–h) and anti-Sca1 (i–l) antibodies. (B) In vitro assay for the early steps of GPI biosynthesis. Cell lysates were incubated with radiolabeled UDP-GlcNAc (upper panel) and with radiolabeled UDP-glucose to assay Dol-P-Glu synthase as a measure of the amount of lysate (lower panel). The lipids were extracted and analyzed by TLC. Identities of spots are indicated on the left. Lane 1, 2.10 GPI (+); lane 2, 2.10 GPI (–); lane 3, 2.10 GPI (–) transfected with pME-neo-PIG-P; lane 4, 2.10 GPI (–) transfected with pME-neo; lane 5, wild-type JY25 cells; and lane 6, mutant JY5 cells. The radiolabeled products above GlcNAc-PI seen in all lanes are non-GPI products because they are seen with cell lysates from JY5 (lane 6), which is known to be deficient in the first step of GPI biosynthesis. (C) RT–PCR analysis of PIG-P in 2.10 GPI (+) and 2.10 GPI (–) cells. Samples of RNA were incubated in the presence (lanes 2 and 4) and absence (lanes 1 and 3) of reverse transcriptase. PCRs were done for the amplification of mouse PIG-P (upper panel) and GPI1 (lower panel).

Fig. 4. Interaction of PIG-P with other components of GPI-GnT. FLAG-PIG-P was co-expressed in JY5 cells with GST–PIG-A (lane 1), –PIG-H (lane 2), –PIG-C (lane 3), –GPI1 (lane 4) or –ALDH (lane 5), and their associations were analyzed by co-precipitation. The lysates (in 1% digitonin) were precipitated with anti-FLAG beads and immunoprecipitates were analyzed by western blotting against anti-FLAG (top panel) and anti-GST (middle) antibodies. GST-tagged proteins were collected from the supernatant obtained after the immunoprecipitation by glutathione beads and analyzed by western blotting against anti-GST antibody (bottom). An asterisk in the middle panel indicates the heavy chain of mouse anti-FLAG antibody that was recognized by protein G. Positions of molecular size markers are indicated on the left (kDa).

Fig. 5. (A) Interaction of DPM2 with GPI-GnT complexes. GST–ALDH (lanes 1 and 4), –DPM2 (lanes 2 and 5) and –DPM1 (lanes 3 and 6) were co-expressed in JY5 cells with the five FLAG-tagged GPI-GnT components (lanes 1–3), or FLAG-DPM3 plus -ALDH (lanes 4–6). Physical associations between GST- and FLAG-tagged proteins were analyzed by co-precipitation. The lysates in 1% digitonin were precipitated with glutathione beads, and precipitates were analyzed by western blotting against anti-GST (top panel) and anti-FLAG (middle) antibodies. FLAG-tagged proteins remaining in the supernatant after precipitation with glutathione beads were immunoprecipitated and analyzed by western blotting against anti-FLAG antibody (bottom). Positions of molecular size markers are indicated on the left (kDa). (B) Association of DPM2 with components of GPI-GnT. GST–DPM2 was co-expressed in JY5 cells with FLAG-ALDH (lane 1), -PIG-A (lane 2), -PIG-H (lane 3), -PIG-C (lane 4), -GPI1 (lane 5) -PIG-P (lane 6) or -DPM3 (lane 7), and their associations were analyzed by co-precipitation. The lysates were precipitated with anti-FLAG and the immunoprecipitates were analyzed by western blotting against anti-FLAG (top panel) and anti-GST (middle) antibodies. GST–DPM2 remaining in the supernatants after immunoprecipitation was assessed by precipitation with glutathione beads and western blotting (bottom). An asterisk in the middle panel indicates a faint band seen in lane 1 showing a background level of GST–DPM2 present in the precipitates.

Fig. 6. GPI-GnT activity of Lec15/B5 cells transfected with various vectors. (A) GPI-GnT (top panel), Dol-P-Man synthase (middle) and Dol-P-Glu synthase (bottom) activities of Lec15/B5 cells transfected with an empty vector (lane 1), DPM1 (lane 2), DPM2 (lane 3), DPM3 (lane 4) or yeast DPM1 (lane 5). Cell lysates were incubated with radiolabeled UDP-GlcNAc (top), GDP-mannose (middle) or UDP-glucose (bottom). The lipids were analyzed by TLC. The identities of spots are indicated on the left. (B) Restoration of the surface expression of GPI-anchored proteins on Lec15/B5 cells transfected with an empty vector (a), DPM1 (b), DPM2 (c), DPM3 (d) or yeast DPM1 (e).