PIG-W is critical for inositol acylation but not for flipping of glycosylphosphatidylinositol-anchor

Abstract

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.

Figure 1.

Defective surface expression of GPI-anchored proteins on Molt4–1D10 and CHOPA10.14 cells. Wild-type Molt4, mutant Molt4–1D10, wild-type CHO-3B2A, and mutant CHOPA-10.14 cells were stained for DAF (top) and CD59 (bottom) and analyzed in a FACScan. Solid lines, specific monoclonal antibodies; dotted lines, isotype-matched control antibodies.

Figure 2.

Accumulation of abnormal, PI-PLC-sensitive GPI species in Molt4–1D10 (A) and CHOPA10.14 (B) cells. (A) Class K mutant K562 (lane 1), wild-type Molt4 (lane 2), and 1D10 mutant (lane 3) cells were cultured with [³H]mannose in the presence of tunicamycin. Mannolipids were analyzed by TLC with a solvent system of chloroform/methanol/H₂O (10:10:3). DPM, dolichol-phosphate-mannose; H5, H6, H7, and H8 are according to Hirose et al., 1992. *a–*f, abnormal mannolipids accumulated in 1D10 cells. Labeled lipids of class K cells (lanes 4, 5, 8, and 9) and 1D10 cells (lanes 6, 7, 10, and 11) were treated with GPI-PLD (lanes 4 and 6), buffer for GPI-PLD (lanes 5 and 7), PI-PLC (lanes 8 and 10), or buffer for PI-PLC (lanes 9 and 11). Reextracted lipids were analyzed by TLC. (B) Mannolipids of CHOPA10.14 mutant cells prepared in a similar way to that described in A were treated with GPI-PLD (lane 1), buffer for GPI-PLD (lane 2), PI-PLC (lane 3), or buffer for PI-PLC (lane 4). *, abnormal mannolipid likely to be Man-GlcN-PI (see RESULTS).

Figure 3.

Characterization of mannolipids accumulated in Molt4–1D10 (A and B) and analysis of early steps in GPI biosynthesis (C). (A) Molt4–1D10 cells cultured in the presence (lane 1) or absence (lane 2) of YW3548/BE49386A for 24 h were metabolically labeled with [³H]mannose. The lipids were analyzed by TLC with a solvent system of chloroform/methanol/H₂O (10:10:3). (B) Molt4–1D10 cells were metabolically labeled with [³H]mannose. Lipids were treated with Jack bean α-mannosidase (lane 1) or buffer (lane 2), extracted again, and analyzed by TLC as described in A. Mannolipids *c and *d were eluted from the TLC plate (lanes 4 and 6) and treated with the α-mannosidase and reanalyzed by TLC (lanes 3 and 5). (C) Wild-type Molt4 (lane 1), 1D10 mutant (lane 2), 1D10 transfected with rat PIG-W cDNA (lane 3), and class E mutant (lane 4) cells were cultured in a medium containing myo-[³H]inositol for 1 d. The radiolabeled lipids were analyzed by TLC with a solvent system of chloroform/methanol/1 M NH₄OH (10:10:3). Lane 5, standard GlcNAc-PI, GlcN-PI, and GlcN-acylPI generated by incubating membranes of wild-type Molt4 with UDP-[6-³H]GlcNAc. PI and GlcNAc-PI are overlapping.

Figure 4.

Molt4–1D10 and CHOPA10.14 cells are defective in PIG-W. (A) Rat PIG-W cDNA restored the surface expression of CD59 on Molt4–1D10 and CHOPA10.14 cells. 1D10 mutant transfected with an empty vector (a) or PIG-W (b) and CHOPA10.14 mutant transfected with an empty vector (c) or PIG-W (d) were stained 2 d after transfection. Solid lines, transfectants; dotted lines, wild-type cells. (B) Normalized GPI biosynthesis in 1D10 cells transfected with PIG-W cDNA. Wild-type Molt4 (lane 1), 1D10 mutant (lane 2), 1D10 transfected with rat PIG-W cDNA (lane 3), and 1D10 mutant transfected with an empty vector (lane 4) were labeled with [³H]mannose and lipids analyzed by TLC.

Figure 5.

(A) Alignment of amino acid sequences of PIG-W homologs of human, rat, S. cerevisiae, and S. pombe. Black and gray boxes indicate identical and conserved amino acids, respectively. Six regions highly conserved in four species are indicated by broken underlines. Positions of 13 predicted transmembrane domains of S. pombe PIG-W are indicated by numbered underlines. An arrowhead indicates leucine changed to a stop codon in 1D10 mutant. (B) Hydropathic profile of human PIG-W. Hydrophobicity was calculated with a window length of 17 (Kyte and Doolittle, 1982). Positions of 13 predicted transmembrane domains are indicated by numbered thick bars.

Figure 6.

ER localization of PIG-W. JY25 cells expressing GST-tagged PIG-W were hypotonically lysed and the postnuclear supernatant fractionated by sucrose density gradient centrifugation. Fractions 1–5 were characterized by protein content (bars) and organelle marker enzymes (alkaline phosphodiesterase I for the plasma membrane, α-mannosidase II for the Golgi apparatus, and dolicholphosphate-mannose synthase for the ER) (A) and by Western blotting with anti-GST antibody (B). The enzyme activities in fractions are shown as percentages of the total.

Figure 7.

CHOPA10.14 and Molt4–1D10 are defective in inositol acylation of a synthetic substrate GlcN-PI(C8). The membranes of wild-type CHO (lanes 1 and 3) and CHOPA10.14 (lanes 2) cells were incubated with [³H]palmitoyl-CoA in the presence (lanes 1 and 2) or absence (lane 3) of GlcN-PI(C8). The membranes of wild-type Molt4 (lane 4), Molt4–1D10 transfected with PIG-W (lanes 5 and 6), and 1D10 mutant (lane 7) were incubated with [³H]palmitoyl-CoA in the presence (lanes 4, 5, and 7) or absence (lane 6) of GlcN-PI(C8). After 10-min-incubation, lipids were analyzed by TLC with a solvent system of chloroform/methanol/0.25% KCl (55:45:10). *, GlcN-acylPI(C8); **, GlcN-PI(C8)-dependent extraspot of unknown structure.

Figure 8.

The PIG-W gene encodes acyltransferase. (A) FLAG-PIG-W-FLAG-HAT (lanes 1 and 4), FLAG-PIG-W-GST (lanes 2 and 5), and PIG-LGST-FLAG (lanes 3 and 6) were isolated by two-step affinity purification from the lysates of transfected JY25 cells. M, molecular size markers; lanes 1–3, silver staining; lanes 4–6, Western blotting with anti-FLAG antibody. (B) The purified FLAG-PIG-W-GST (lanes 1 and 3) and PIG-L-GST-FLAG (lane 2) proteins were incubated with [³H]palmitoyl-CoA in the presence (lanes 1 and 2) or absence (lane 3) of GlcN-PI(C8). After 10-min-incubation, lipids were analyzed by TLC with a solvent system of chloroform/methanol/0.25% KCl (55:45:10). GlcN-acylPI(C8) generated (asterisk) was treated overnight with GPI-PLD (lane 4), buffer for GPI-PLD (lane 5), PI-PLC (lane 6), or buffer for PI-PLC (lane 7), extracted again, and analyzed by TLC.

Figure 9.

Membrane orientation of the N and C termini of PIG-W. Rat PIG-W tagged at the C termini (PIG-W-FLAG) (a–f) or the N terminus (FLAG-PIG-W) (g–l) were expressed in CHO cells. After permeabilization of both the plasma membrane and the ER membrane (a–c and g–i), or selective permeabilization of the plasma membrane (d–f and j–k), cells were stained for tagged PIG-W by anti-FLAG antibody (b, e, h, and k), for an endogenous ER luminal protein BiP (a, d, g, and j) and a cytosolic protein Hsc70 (c, f, I, and l).