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Fig. 1. GMT recycles between the ER and Golgi apparatus. (A,B) Yeast cells producing the Myc₆-tagged GMT, Anp1p or Van1p from CEN plasmid were fixed with formaldehyde at the time indicated after the shift to 37°C and prepared for indirect immunofluorescence staining using anti-Myc monoclonal antibody 9E10 and FITC-conjugated affinity-purified goat anti-mouse-IgG. Cells were treated with cycloheximide (0.1 mg ml^–1) before being shifted to 37°C. After 20 minutes at 37°C, the culture was divided in two; one portion was kept at 37°C and the other was shifted back to 25°C and incubated for 20 minutes to see the reversibility of localization (Recovery). (A) Wild-type (WT) and sec18^ts mutant cells expressing Myc₆-GMT. (B) Wild-type and sec23^ts mutant cells expressing Myc₆-GMT, Myc₆-Anp1p and Myc₆-Van1p. (C) Subcellular fractionation of the wild type and sec23-mutant-expressing Myc₆-GMT. Medium-speed pellet (P7), high-speed pellet (P100) and high-speed supernatant (S100) were prepared, and GMT was detected by immunoblotting.

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