Figure 9.

Nce102-GFP localization depends on sphingolipid levels. (a) Nce102-GFP was imaged under normal growth conditions (control), after addition of 5 µM myriocin for 1 h (myriocin), after sequential treatment with 5 µM myriocin for 1 h and 50 µM PHS for 15 min (myriocin→PHS), or after addition of 50 µM PHS for 15 min (PHS). Boxes indicate the area magnified in the bottom panels. (b) Fluorescence intensities of the area are shown plotted against xy image coordinates. (c) Nce102 redistribution is not dependent on new protein synthesis. Nce102-GFP cells were treated with myriocin or myriocin and PHS successively as in a after 10-min preincubation and continued presence of cycloheximide (CHX). (d) Nce102 partitions into detergent-resistant membranes dependent on sphingoid bases. Untreated Nce102-TAP–expressing cells and cells treated as in a were lysed in buffer containing 1% Triton X-100 and analyzed by gradient centrifugation and Western blotting against TAP (left). The same blots were probed with Pma1 antibodies (right). (e) pil1(4A)-GFP is resistant to disassembly after myriocin treatment. pil1(4A)-GFP–expressing cells were imaged after 1-h 5 µM myriocin incubation. (f) Redistribution of Nce102-GFP after myriocin treatment is independent of eisosome disassembly. Cells expressing pil1(4A) and Nce102-GFP were imaged before (left) and after 1 h treatment with 5 µM myriocin (right). (g and h) Sur7-mars does not behave like Nce102 after myriocin treatment. Wild-type Pil1 (g) or pil1(4A) (h) cells expressing Sur7-mars were treated with myriocin and imaged. wt, wild type. Bars, 5 µm.