Figure 7.

A functional Golgi complex is essential for autophagy. (A) The sec12 (FBY217) and sec7 (AFM69-1A) strains were transformed with both a multi-copy plasmid expressing Ape1 and either an empty vector (pRS415) or one carrying the wild-type allele of the mutated gene (pSEC12 or pSEC7, respectively). Transformed cells and two wild-type strains (WT, SEY6210, and BY4742) were grown to 1 OD₆₀₀ in SMD medium at 24°C. After washing twice with water, cells were nitrogen starved in SD-N medium for 4 h at 37°C before collecting the proteins by TCA precipitation. Polypeptides were finally resolved by SDS-PAGE and prApe1 maturation analyzed by immunoblot with serum to Ape1. (B) The sec12 (FRY208) and sec7 (FRY209) strains expressing Pho8Δ60 and carrying either an empty vector (pRS415) or a plasmid complementing the corresponding sec mutation (pSEC12 or pSEC7, respectively) were shifted from SMD medium at 24°C (black bars) to SD-N medium at 34°C (white bars) for 4 h. Autophagy induction was determined by a Pho8 activity assay. Results were expressed as percentage of the activity measured for the complemented strains starved for nitrogen.