Figure 9.
Sec7 is essential for the phagophore expansion. Wild-type (strain AVY047; A) and sec7ts (strain AVY048; B) strains expressing RFP-Ape1 and carrying the pCuGFPATG8416 plasmid were grown at 24°C to an early log phase. Rapamycin was then added to the cultures, which were kept either at 24°C or transferred to 37°C for 1 h. Cells were finally visualized by fluorescence microscopy. The prApe1 oligomer is associated to the PAS in the sec7ts mutant at restrictive temperatures. (C–F) The sec7ts (AFM69-1A) strain was grown at 24°C to an early log phase before being transferred to 37°C for 1 h in the SD-N medium. Cells were processed for IEM as described in Materials and Methods and the PAS identified by labeling the cryosections with an anti-Ape1 antibody followed by protein A–15-nm gold particle conjugates. V, vacuole. Bar, 200 nm. DIC, differential interference contrast.