Figure 4.

Ceramide treatment restores the degradation of Ste2 in snc and temperature-shifted snc1^ala43 cells. snc (JG8) and snc1^ala43 (JG8-SNC1A43) cells expressing Ste2HA were grown to log phase in glucose-containing medium at 26°C with (+C₂) or without (−C₂) C₂-ceramide. snc cells expressing SNC1 constitutively (SNC1) were grown without ceramide and were used as control. Cells were pulse-labeled with [³⁵S]methionine and chased for 30 min in medium containing 5 mM methionine/cysteine, before treatment with α-factor (1 μM). For snc1^ala43 cells, half of the cultures were shifted to prewarmed medium (37°C) for 1 h before labeling. At various times after treatment with α-factor (2–60 min), samples were removed, extracts were prepared, and Ste2HA was IP'd using anti-HA antibodies. Ste2HA was detected by autoradiography.