Figure 1.

TORC2-Ypk1 signaling regulates ATG8 expression via the heterodimeric zinc-finger transcription factor Msn2-Msn4. (A) Schematic representation of the ATG8 promoter region, indicating the identified recognition site for the HDAC subunit Ume6²⁵ as well as potential stress response elements (STRE) as determined by YEASTRACT.³⁰ Also indicated are positions of G to C mutations introduced into the STRE sites. (B) Indicated strains carrying pRS416 prATG8-GFP-ATG8 were grown to log phase in SCD medium without uracil. GFP-Atg8 protein was visualized via western blot analysis as described in Materials and Methods. GFP-Atg8 protein expression was normalized to the Zwf1 loading control and quantification represents Atg8 protein levels relative to WT, presented as the mean ± standard deviation (SD) of at least 3 independent experiments. (C) Indicated strains expressing endogenously tagged MSN2-GFP were visualized by fluorescence microscopy as described in Materials and Methods. Scale bar: 5 μm. ((D)and E) Indicated strains carrying pRS416 prATG8-GFP-ATG8 or pRS416 prATG8-GFP-ATG8 possessing STRE mutations depicted in (A) were grown to log phase in SCD medium without uracil. GFP-Atg8 protein levels were detected by western blot and analyzed as described in (B).