Figure 7.

Modification of C₆-NBD-ceramide by living yeast cells. (A) TLC separation of the products of lipids C₆-NBD-ceramide extracted either from microsomes (memb), as in Figure 4, or from live yeast incubated with C₆-NBD-ceramide, as described in MATERIALS AND METHODS. AbA treatment was at 10 μg/ml, with a 10-min preincubation for the cells. The faster migrating band is the precursor C₆-NBD-ceramide. (B) As in A except that wild-type (SEY6211; wt) and sec6-4 (JWY47; sec6) cells were incubated with C₆-NBD-ceramide for 10 min at 25°C and then for 5 min at 37°C. After washing and back-extraction for 60 min on ice, cells were resuspended into fresh, prewarmed medium containing 10 mg/ml defatted BSA and 10 μg/ml AbA and incubated for 10 min at 37°C, and lipids were extracted from cells and medium. (C) Live yeast labeled with C₆-NBD-ceramide with or without AbA, as described in MATERIALS AND METHODS, and photographed with both fluorescence and Nomarski optics. Rings can be seen around the nucleus (n) distinct from the vacuole (v). Bar, 1.5 μm.