Characterization of GAA1-knockout cells. (A) Surface expression of GPI-anchored proteins. Parent F9 cells (a) and GAA1-knockout cells (b) were analyzed for the surface expression of Thy-1. GAA1-knockout cells transiently transfected with an empty vector (c) or human GAA1 cDNA (d) were stained for Thy-1 2 d after transfection. Solid and dotted lines indicate staining with anti–Thy-1 antibody and that with secondary reagent alone, respectively. (B) Biosynthesis of GPI anchor intermediates analyzed by TLC. Cells were metabolically labeled with [³H]mannose for 45 min in the presence of tunicamycin, and mannolipids were analyzed by TLC and fluorography. Identities of spots and origin are indicated on the right. DPM, dolichol–phosphate-mannose; M2 and M3, GPI intermediates bearing two and three mannose residues, respectively; H7 and H8, mature forms of GPI. Lanes 1 and 2, class B and F GPI-deficient mutants used to help identify the spots; lanes 3 and 4, wild-type and class K mutant of K562 cells; lanes 5 and 6, GAA1-knockout and wild-type F9 cells.

Cysteine and histidine residues conserved in Gpi8ps and cysteine proteases are essential for Gpi8p activity. (A) Alignment of partial sequences of human Gpi8p, yeast Gpi8p, nematode (Caenorhabditis elegans) Gpi8p (T05E11.6) (Wilson et al., 1994), and two proteases belonging to a novel cysteine protease family from Canavalia ensiformis (Abe et al., 1993), Citrus sinensis (Alonso and Granell, 1995) using Clustal W software. Amino acids of human Gpi8p substituted for alanine are marked with asterisks. (B) Activities of alanine mutants of human Gpi8p. GST-tagged wild-type and mutant human GPI8 cDNAs were transiently transfected into class K cells. Cells were stained for CD59 and analyzed by flow cytometry 2 d after transfection. Solid and dotted lines indicate staining with anti-CD59 antibody and that with secondary reagent alone, respectively. (C) Expression of mutant Gpi8ps in the transfectants used in B. GST-tagged Gpi8ps in detergent extracts of the transfectants were collected with glutathione beads and analyzed by Western blotting with anti-GST antibody. Molecular markers in kilodaltons are indicated on the left. (D) In vitro assay with mini-PLAP. Microsomal membranes of class K cells stably expressing GST-tagged wild-type or mutant human Gpi8p were tested for the formation of carbonyl intermediates of mini-PLAP in vitro in a similar way as in Figure 3. Molecular markers in kilodaltons are indicated on the left.

Association of human Gaa1p and Gpi8p shown by coimmunoprecipitation. Plasmids for FLAG- or GST-tagged proteins were transiently transfected into CHO cells in the indicated combinations. NP-40 extracts were prepared 2 d later and were first incubated for precipitation with anti-FLAG beads. The precipitates were analyzed by Western blotting with anti-FLAG (lanes 1–3) and anti-GST (lanes 4–6) antibodies. The supernatants of the first precipitation were then subjected to precipitation with glutathione beads and Western-blotted with anti-GST antibody (lanes 7–9). Double asterisks indicate the heavy chain of anti-FLAG antibody, and single asterisks indicate nonspecific bands of unknown origin (lanes 4–6). Molecular markers in kilodaltons are indicated on the left.

Generation of mouse GAA1-knockout cells. (A) Structure of the mouse GAA1 gene (top), targeting constructs (middle), and expected disrupted alleles (bottom). Four consecutive recombination-targeting constructs in which drug resistance genes (drug^r) were neomycin (neo), puromycin (puro), hygromycin (hyg), and blasticidin (bsd) resistance genes, respectively, were used in this order. The mouse GAA1 gene consists of 12 exons (shown in boxes: open areas, coding regions; closed areas, noncoding regions). Closed arrows represent PCR primers for screening recombinants. Probes for Southern analysis are indicated by thick bars. Predicted sizes of EcoRV- and EcoRI-digested fragments hybridized with 5′ and 3′ probes, respectively, are indicated above each allele. RV, EcoRV; RI, EcoRI; B, BamHI; X, XbaI; S, SacII (X and S are not unique). (B) Southern analysis of GAA1-targeted F9 cells. EcoRV-digested (left) and EcoRI-digested (right) genomic DNA from wild-type cells (W) and cells after single (S), double (D), triple (T), and quadruple (Q) recombinations were hybridized with the 5′ probe (left) and 3′ probe (right), respectively. The sizes of hybridized bands are indicated on the right.

Microsomal membranes from GAA1-knockout cells do not form a carbonyl intermediate of mini-PLAP. Capped mini-PLAP mRNA was translated in vitro using rabbit reticulocyte lysate and microsomal membranes prepared from the indicated cells in the absence (− or presence (+) of hydrazine. Lane 1 in both A and B shows a translation product in the absence of microsomal membranes, yielding prepro–mini-PLAP. Identities of other bands determined according to Kodukula et al. (1991) are shown on the right. Positions of prestained molecular markers are shown on the left. The cellular origin of the microsomal membranes is indicated as well as the presence or absence of hydrazine. In B, lanes 6 and 7 show the microsomal membranes from GAA1-knockout cells rescued with human GAA1 cDNA, and lanes 8 and 9 show those from GAA1-knockout cells transfected with human GPI8 cDNA.

Conserved cysteine and histidine residues in yeast Gpi8p are essential for function. (A) Schematic representation of human and yeast Gpi8ps: hatched, dotted, cross-hatched, closed, and open boxes correspond to segments containing amino-terminal signal sequence, highly conserved, nonconserved, transmembrane (TM), and cytoplasmic regions, respectively. The two proteins were divided into four segments, R1–R4, and chimeric molecules were generated. The values in parentheses are percentage amino acid identity between the corresponding regions. Amino acid numbers and the positions of important histidine and cysteine residues are indicated. (B) Activities of a chimeric Gpi8p consisting of human R1, yeast R2, and human R3 and R4, and its alanine mutants. Chimeric Gpi8p and its mutants were transiently transfected into class K cells, and surface expression of CD59 was measured by flow cytometry. Solid and dotted lines indicate staining with anti-CD59 antibody and that with secondary reagent alone, respectively.

Lumenal portion of human Gpi8p is sufficient for its functions. (A) Schematic representation of wild-type and carboxyl-terminal deletion mutants of human Gpi8p. 310del, 321del, and 332del refer to deletion mutants of GPI8 encoding amino acids 1–310, 1–321, and 1–332, respectively. Segments are patterned in the same way as in Figure 5A. (B) Wild-type and deletion mutants of human GPI8 were transiently transfected into class K cells. Surface expression of CD59 was measured by flow cytometry 2 d later. Solid and dotted lines indicate staining with anti-CD59 antibody and that with secondary reagent alone, respectively. (C) Association of HA-tagged Gaa1p and FLAG-tagged deletion mutants of Gpi8p were analyzed by coprecipitation. Plasmids for HA- or FLAG-tagged proteins were transiently transfected into CHO cells in the indicated combinations. The precipitates from NP-40 lysates with anti-HA antibody were subjected to Western blotting with anti-HA (lanes 1–6) or anti-FLAG (lanes 7–12) antibodies. The remaining supernatants were then subjected to precipitation and blotted with anti-FLAG antibody (lanes 13–18). An asterisk indicates nonspecific bands of unknown origin that overlap with a band of 310del Gpi8p (lane 15). Molecular markers in kilodaltons are indicated on the left.