Fig. 1. Identification of two new components of GPI transamidase. The transamidase complex was purified from the NP-40 extract of class K cells stably expressing FLAG-GST-tagged GPI8 by two-step affinity purification. A sample of protein complex (equivalent to 2 × 10⁸ cells) was separated on a 10% SDS–polyacrylamide gel under reducing conditions and silver stained (lane 2). Bands were numbered 1–4 from top to bottom. The N-terminal amino acid sequences (X is an unidentified amino acid) and protein identities are indicated on the right. Lane 1, molecular size markers.

Fig. 2. (A) Alignment of protein sequences of PIG-S homologues. Human PIG-S (accession No. AB057723) and its S.cerevisiae (S69714) and S.pombe (T38067) homologues are aligned using the Multiple Alignment Program software (Huang, 1994). The human PIG-S sequence determined by N-terminal sequencing is overlined. Black and grey boxes are identical and similar amino acids, respectively. (B) Hydrophobicity profile of human PIG-S protein according to the Kyte and Doolittle method (Kyte and Doolittle, 1982). Putative transmembrane domains near the N- and C-termini are indicated by thick bars.

Fig. 3. (A) Alignment of protein sequences of PIG-T homologues. Human PIG-T (AB057724) and its S.cerevisiae (S46687) and S.pombe (T39499) homologues are aligned using the Clustal W software. The human PIG-T sequence determined by N-terminal sequencing is overlined. Di-lysine ER retention signals in S.cerevisiae (KXKXX) and S.pombe (KKXX) are marked by open and closed triangles, respectively. (B) Kyte and Doolittle hydrophobicity plot of human PIG-T protein. The N-terminal signal peptide and a putative transmembrane domain near the C-terminus are indicated by thick bars.

Fig. 4. Generation of PIG-S and PIG-T knockout mouse F9 cells. (A) Disruption of mouse PIG-S (upper) and PIG-T (lower) genes. Structures of the mouse PIG-S and PIG-T genes (top), targeting constructs (middle) and expected mutant alleles (bottom) are shown. Exons are indicated as boxes (closed boxes, coding regions; open boxes, untranslated regions). Exon 1 of the PIG-S gene is located 1.6 kb downstream to exon 8 of the aldolase C gene. First and second round disruptions of both PIG-S and PIG-T were performed with targeting constructs bearing puromycin and neomycin resistance genes, respectively. Thin arrows represent PCR primers for screening the recombinants. Probes for Southern analysis are indicated by thick bars. The predicted sizes of fragments hybridized with 5′ and 3′ probes are indicated above each genomic structure. A, ApaI; H, HindIII; N, NotI; K, KpnI; RI, EcoRI; RV, EcoRV; S, SpeI; Xb, XbaI; Xh, XhoI (italicized sites in the figure are not unique). (B) Southern analysis of PIG-S and PIG-T knockout F9 cells. Digested genomic DNA from wild-type cells (F9), heterozygous (Hetero) and homozygous (Homo) knockout cells for PIG-S (left panels) and PIG-T (right panels) genes were hybridized with the 5′ probe (top) and 3′ probe (bottom) shown in (A). Hybridized fragments of wild-type (W) and mutant (M) alleles, and size markers are indicated on the right and left, respectively. Enzymes used for digestion are shown below each panel.

Fig. 5. PIG-S and PIG-T are essential components of GPI transamidase. (A) Parent F9 cells, and PIG-S and PIG-T knockout cells transfected with an empty vector or corresponding human cDNA were analysed for the surface expression of Thy-1 by flow cytometry 2 days after transfection. Solid and dotted lines indicate staining with anti-Thy-1 antibody and with secondary reagent alone, respectively. (B) Biosynthesis of GPI anchor intermediates. Cells were metabolically labelled with [³H]mannose for 60 min in the presence of tunicamycin. Mannolipids were extracted and analysed by TLC. Identities of spots according to Hirose et al. (1992) and origin are indicated on the right. H5 and H6, GPI bearing two and three mannoses, respectively; H7 and H8, mature forms of GPI. (C) In vitro assay for GPI transamidase. MiniPLAP mRNA was translated in vitro using rabbit reticulocyte lysates and the microsomal membranes prepared from the indicated cells in the absence (–) or presence (+) of hydrazine. The class F cell is defective in biosythesis of GPI. The class K cell is a mutant of GPI8. The translation product in the absence of the microsomal membranes is prepro-miniPLAP (lane 1). Identities of other bands according to Kodukula et al. (1991) are shown on the right. Schematic representations of the products are shown below.

Fig. 6. Saccharomyces cerevisiae GPI16 and GPI17 are orthologues of PIG-T and PIG-S, respectively. Wild-type, and gaa1, gpi8, gpi16 and gpi17 deletant haploid yeast cells bearing a GAL1-regulatable expression plasmid for the respective ORF were pre-cultured in 2% galactose (for gaa1, gpi8 and gpi16 deletants) or 2% raffinose (for the gpi17 deletant) and transferred to 5% glucose-containing medium. gaa1, gpi8, gpi16 and gpi17 deletants were metabolically labelled with [³H]inositol at 24, 12, 12 and 24 h, respectively, after transfer. Mannolipids were extracted and analysed by TLC. The identities of spots (Hamburger et al., 1995; Benghezal et al., 1996) are indicated on the left. Four lines analysed in groups were derived from the same ascus. W, wild type; M, mutant.

Fig. 7. PIG-T is critical for formation of the GPI transamidase complex. The four differentially tagged transamidase components and ALDH were transfected into CHO cells in various combinations (combination of transfection shown above panels). Cells were lysed in a buffer containing 1% NP-40 2 days after transfection. Aliquots of the lysates were immunoprecipitated (IP) individually with one of the five antibodies as indicated and western blotted against a mixture of five antibodies. Identities of bands are on the left. Size markers in kDa are on the right. (A) Combinations of all four (lanes 1–5) and three out of four (lanes 6–25) components. In lanes 21–23, bands between HA-PIG-S and FLAG-GAA1 are not GST–GPI8; they are derived from HA-PIG-S and migrate slightly more slowly than GST–GPI8. (B) Pairwise combinations of PIG-T and GAA1 (lanes 1–5) or GPI8 (lanes 6–10). (C) A combination of PIG-S and PIG-T.

Fig. 8. (A) Membrane topology of GPI transamidase components. N- and C-termini are shown. GAA1 has a large luminal domain and multiple transmembrane regions. Both N- and C-termini of GAA1 are predicted to be on the cytoplasmic side (Hamburger et al., 1995). PIG-S has two transmembrane regions near the N- and C-termini. GPI8 (Benghezal et al., 1996) and PIG-T are type-I transmembrane proteins. (B) A model of GPI transamidase. The GPI transamidase is a complex consisting of at least four components. PIG-T is a central component interacting with GAA1, GPI8 and PIG-S. The catalytic component, GPI8, recognizes substrate proproteins and attacks a peptide bond between ω and ω+1 sites with a sulfhydryl group of its active site cysteine to form a carbonyl intermediate. It is unclear how GPI is presented to the carbonyl intermediate to complete transamidation.