Figure 6. Comparison of methods for inactivating Sec21.
General secretion was visualized by a pulse-chase analysis as in Figure 5A, except that cells were grown at 23°C and then shifted to 37°C for 30 min before the procedure. A control strain expressed the mitochondrial OM45-FKBPx4 anchor. Where indicated, a strain expressed OM45-FKBPx4 plus either Sec31-FRBx2 to anchor COPII or Sec21-FRBx2 to anchor COPI. The sec21-3 mutation was in the parental strain background. “Rap” indicates a 10-min treatment with 1 μg/mL rapamycin prior to the pulse. Numbers represent the molecular weight in kDa of marker proteins.
Figure 6—figure supplement 1. Thermosensitivity of the sec21-3 strain.
Ten μL of 10-fold serial dilutions of wild-type or isogenic sec21-3 mutant cells were spotted in parallel on two YPD plates. The plates were then incubated for two days at either 23°C or 37°C.
Figure 6—figure supplement 2. Perturbed Golgi structure in sec21-3 cells at the permissive temperature.
A wild-type strain or an isogenic sec21-3 strain were transformed to express the early Golgi marker GFP-Vrg4 and the late Golgi marker Sec7-DsRed. Cells were imaged at 23°C by widefield microscopy. Representative cells are shown. Scale bar, 2 µm.