Figure 2. The emp24Δ ret4-1 double... | Rockefeller University Press

Figure 2.

$Figure 2. The emp24Δ ret4-1 double mutant cells exhibit COPI mutant–like phenotypes. (A) emp24Δ strain was crossed with a ret4-1 strain and the resulting haploid spores were tested for growth at 24 and 35°C on YPUAD. (B) Proliferating cells were radiolabeled for 5 min, chased for 30 min at 24°C, and lysed. CPY was immunoprecipitated, resolved by SDS-PAGE, and analyzed by PhosphoImager. ER (p1), Golgi (p2), and vacuole (m) CPY forms are indicated. (C) Low glucose–induced cells were pulse-labeled for 5 min and chased for 30 min at 24°C. Cells were converted to spheroplasts, and separated into intracellular (I) and extracellular (E) fractions from which invertase was immunoprecipitated, resolved by SDS-PAGE, and analyzed by PhosphoImager. Migration positions of core glycosylated, hypo-, and hyperglycosylated invertase are indicated. (D) Cells without (1, 2, 3, and 4) or with (5, 6, 7, and 8) ERD2 2μ plasmid (pJS209) were transferred to fresh media for 1 h at 24°C. Proteins contained in the cell culture supernatant were concentrated by TCA precipitation, resolved by SDS–PAGE, and Kar2p and Hexokinase were detected by immunoblotting. (E) β-Galactosidase assays were performed on strains harboring the reporter construct (pJC31). (F) Fluorescence images of cells expressing the ER-localized protein GFP-HDEL. Cells were grown at 24°C, except sec21-1 cells, which were grown at 24°C and then shifted 40 min at 37°C. Bar, 5 μm.$

The emp24Δ ret4-1 double mutant cells exhibit COPI mutant–like phenotypes. (A) emp24Δ strain was crossed with a ret4-1 strain and the resulting haploid spores were tested for growth at 24 and 35°C on YPUAD. (B) Proliferating cells were radiolabeled for 5 min, chased for 30 min at 24°C, and lysed. CPY was immunoprecipitated, resolved by SDS-PAGE, and analyzed by PhosphoImager. ER (p1), Golgi (p2), and vacuole (m) CPY forms are indicated. (C) Low glucose–induced cells were pulse-labeled for 5 min and chased for 30 min at 24°C. Cells were converted to spheroplasts, and separated into intracellular (I) and extracellular (E) fractions from which invertase was immunoprecipitated, resolved by SDS-PAGE, and analyzed by PhosphoImager. Migration positions of core glycosylated, hypo-, and hyperglycosylated invertase are indicated. (D) Cells without (1, 2, 3, and 4) or with (5, 6, 7, and 8) ERD2 2μ plasmid (pJS209) were transferred to fresh media for 1 h at 24°C. Proteins contained in the cell culture supernatant were concentrated by TCA precipitation, resolved by SDS–PAGE, and Kar2p and Hexokinase were detected by immunoblotting. (E) β-Galactosidase assays were performed on strains harboring the reporter construct (pJC31). (F) Fluorescence images of cells expressing the ER-localized protein GFP-HDEL. Cells were grown at 24°C, except sec21-1 cells, which were grown at 24°C and then shifted 40 min at 37°C. Bar, 5 μm.