Figure 3.

Influence of secretory cargo on tER site morphology. (A) Wild-type cells expressing Sec13p-GFP (CBY1829) were treated with 8 mM DTT or 0.1 mg/ml cycloheximide for indicated times. Cells were collected and whole cell lysates assessed for CPY and actin (loading control) by immunoblot. Note accumulation of p1 CPY (ER form) in DTT-treated cells and depletion of p1 CPY in cycloheximide-treated cells compared with wild type. (B) sec12-4 (CBY1772) and sec16-2 (CBY1999) cells expressing Sec13p-GFP were grown at 20°C, treated with 0.1 mg/ml cycloheximide for 30 min, and then shifted to 37°C. Cells were imaged at 0-, 10-, 30-, and 60-min time points after temperature shift, and images are shown at the 30-min time point (1 h after receiving cycloheximide). (C) Quantification showing the percentage of cells with altered tER site morphology from experiment shown in B. At least 150 cells were counted per condition. (D) Wild-type cells expressing Sec13p-GFP (CBY1829) were grown to mid-logarithmic phase, treated with 8 mM DTT, and imaged after 1 h. (E) Total fluorescence intensity per tER site was analyzed in 10 randomly selected cells per condition, from the images shown in D.