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).