Figure 1.—

Activation of the UPR in cells blocked in secretion and vacuolar targeting. (A) Transcription of UPRE is induced in mutants defective in membrane trafficking and transport. CKY8 (wt), RSY263 (sec12^ts-4), CKY46 (sec13^ts-1), CKY49 (sec13^ts-4), RSY281 (sec23^ts-1), RSY267 (sec16^ts-2), RSY269 (sec17^ts-1), RSY271 (sec18^ts-1), RSY279 (sec22^ts-3), RCY927 (sec21^ts-1), RCY243 (sec1^ts-1), RCY248 (sec4^ts-8), JBY318 (sec6^ts-4), RCY260 (sec15^ts-1), and BJY8928 (pep12Δ) were transformed with pJC104 (UPRE-CYC-lacZ). Transformants were precultured at 20° to midlogarithmic phase of growth in medium lacking uracil and shifted to indicated temperatures. Samples were taken after 3 hr following the shift and analyzed for β-galactosidase activity as described in materials and methods. β-Galactosidase activity unit was defined as OD₄₂₀/min/ml. (B) Transcription of UPRE is induced at 30° in BFA-treated erg6Δ cells. HCY 104 (wt) and HCY362 (erg6Δ) carrying pJC104 (UPRE-CYC-lacZ) were grown in uracil dropout medium containing 0, 10, 25, and 50 mm BFA. Samples were taken at 2 hr. β-Galactosidase activity unit was defined as OD₄₂₀/min/ml. (C) Splicing of HAC1 transcripts is elevated in sec13^ts-1 cells. Northern blot analysis was performed to monitor splicing of HAC1 mRNA in sec13^ts-1 cells (CKY49) grown at 25° and 30°. As a control, splicing of HAC1 mRNA was also monitored in wild-type cells (CKY8) treated with 1 mm tunicamycin (Tm). Cells were precultured overnight in YEPD medium at 25° and shifted to indicated temperatures (for wild-type positive control, 1 mm Tm was added). Samples were taken after 3 hr following the temperature shift. ACT1 mRNA is shown as a loading control.