Figure 10.

Formation of lipid 11-1 is dependent on YLL031c but can occur in the absence of GPI7. (A) The YLL031c:: KAN^R- pGAL-YLL031c/gpi11::LEU2-pPIG-F strain was maintained on SGlyYE medium, then shifted to SGlcYE medium for 16 h at 25°C. Cells were then resuspended and incubated in inositol-free medium containing glucose for 30 min at 25°C, after which equal portions were shifted to 37°C for 20 min or maintained at 25°C and then labeled for 2 h with [³H]inositol at 37 or 25°C respectively (lanes 3 and 4, Δ031Δgpi11), as detailed in MATERIALS AND METHODS. The gpi11::LEU2-pPIG-F (lane 5) and gpi7::LEU2 (lane 6) strains were pulse labeled with [³H]inositol at 37°C. A YLL031c::KAN^R- pGAL-YLL031c strain (lane 1) and a YLL031c⁺ sibling of that strain harboring pGAL-YLL031c (lane 2, WT) were grown and labeled as described for Figure 9A, lane1. Radiolabeled lipids were separated by TLC using solvent B. The species indicated by an asterisk is not seen reproducibly, nor is its presence correlated with a deficiency in any single Gpi protein. (B) The gpi7::URA3/gpi11::LEU2-pPIG-F strain was pulse labeled at 37°C with [³H]inositol, and radiolabeled lipids were separated by TLC using solvent B (lane 2). Samples of [³H]inositol-labeled lipids that accumulate in the gpi7::LEU2, gpi11::LEU2-pPIG-F and YLL031c::KAN^R-pGAL-YLL031c strains were loaded in lanes 1, 3, and 4 respectively.