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. 2002 May;13(5):1594–1607. doi: 10.1091/mbc.01-11-0541

Figure 3.

Figure 3

Ligand-mediated delivery of Ste2 to the vacuole is restored in snc, snc tlg, and temperature-shifted snc1ala43 cells by CAPP activation. (A) Delivery of Ste2-GFP, but not Ste2-tailGFP, to the vacuole is restored in ceramide-treated snc cells. snc cells (JG8) expressing either Ste2-GFP (snc STE2GFP) or a truncated form of Ste2-GFP (snc STE2-tailGFP), which does not undergo endocytosis, were grown to log phase in galactose-containing medium at 26°C, before being shifted to glucose-containing medium with (+C2) or without (−C2) added C2-ceramide (10 μM) for 36 h, and before labeling with FM4-64. After labeling, cultures were split, whereby half was mounted in soft agar containing no additions, whereas the other half was mounted in agar containing α-factor (1 μM) and processed immediately for microscopy. snc cells expressing SNC1 constitutively (SNC1 STE2GFP) were used as control. Cells were monitored by confocal microscopy as a function of time. Separate untreated cells (untreated) were used as controls. (B) Delivery of Ste2-GFP to the vacuole is restored in ceramide-treated snc tlg cells. snc tlg1 (JG9-TLG1) and snc tlg2 cells (JG9-TLG2) expressing Ste2-GFP (snc tlg1 STE2GFP or snc tlg2 STE2GFP) from the kanr plasmid were grown in galactose-containing medium at 26°C, before being shifted to glucose-containing medium with (+C2) or without (−C2) C2-ceramide (10 μM). After 24 h, cells were labeled with FM4-64, treated with α-factor and processed for microscopy. (C) Ligand-mediated delivery of Ste2-GFP to the vacuole is restored in snc cells bearing a mutation in VBM1. snc vbm1 cells (MM1) expressing either Ste2-GFP (snc vbm1 STE2GFP) or the truncated form of Ste2-GFP (snc vbm1 STE2-tailGFP) were grown to log phase at 26°C in glucose-containing medium, before labeling with FM4-64, treatment with α-factor, and processing for microscopy. (D) Ligand-mediated delivery of Ste2-GFP to the vacuole occurs in temperature-shifted snc1ala43 cells upon ceramide treatment. snc1ala43 cells (JG8-SNC1A43L) expressing Ste2-GFP were grown at 26°C in glucose-containing medium with (+C2) or without (−C2) C2-ceramide (10 μM). Half of each culture was shifted to prewarmed medium (37°C), for 1 h before FM4-64 labeling and treatment with α-factor at the restrictive temperature.