Figure 2. Complementation of the S. cerevisiae autophagy mutants with S. macrospora autophagy genes. Complementation of respective yeast strains with Smatg8 and Smatg4 was analyzed using a precursor Ape1 maturation assay.³⁷ In S. cerevisiae the cytosolic 61-kDa precursor of Ape1 (prApe1) is delivered to the vacuole via the Cvt pathway, which involves intact Atg4 and Atg8 proteins. Upon delivery to the vacuole prApe1 is processed to the mature Ape1 enzyme (Ape1). Identical amounts of yeast cell extracts (0.2 OD₆₀₀ equivalents of cells) isolated before (-) and after starvation on SD-N medium for 4 h (+) were separated on 15% SDS-PAGE gels. After blotting, PVDF membranes were probed with an anti-Ape1 antibody as stated in the Materials and Methods. To verify equal protein concentrations after stripping of the membrane anti-actin antibody was used as loading control. Numbers at the right indicate the molecular mass. prApe1, precursor Ape1, Ape1, mature Ape1. (A) Complementation of S. cerevisiae atg8∆ with Smatg8 under control of the yeast MET25 promoter (pRSmet-Smatg8). As a positive control the S. cerevisiae wild-type strain WCG was transformed either with the empty plasmid pRS426-met25, or S. cerevisiae atg8∆ was transformed with the yeast egfp-ATG8 gene (pRS315-EGFP-Atg8). S. cerevisiae atg8∆ transformed with the empty vector pRS426-met25 served as a negative control. (B) Complementation of S. cerevisiae atg4∆ with Smatg4 under control of the yeast MET25 promoter (pRSmet-Smatg4). As positive control, the S. cerevisiae wt WCG was transformed with the empty plasmid pRS426-met25 or S. cerevisiae atg4∆ was transformed with the yeast ATG4 gene (pRS316-Atg4). S. cerevisiae atg4∆ transformed with the empty vector pRS426-met25 served as a negative control.