Spontaneous mutations in CYC8 and MIG1 suppress the short chronological lifespan of budding yeast lacking SNF1/AMPK. Saccharomyces cerevisiae

Chronologically aging yeast cultures are prone to adaptive regrowth, whereby mutants with a survival advantage can spontaneously appear and re-enter the cell cycle during stationary phase. Adaptive regrowth is especially noticeable with short-lived strains, including those defective for SNF1, the homolog of mammalian AMP-activated protein kinase (AMPK). SNF1 becomes active in response to multiple environmental stresses that occur during stationary phase, including glucose depletion and oxidative stress. SNF1 is also required for the extension of CLS by caloric restriction (CR), which is defined as glucose restriction at the time of culture inoculation. Under CR conditions, we took advantage of the increased adaptive regrowth frequency in a snf1∆ mutant to screen for long-lived snf1∆ suppressor mutants from aging cultures and identify the SNF1-regulated processes most important for maintaining CLS. Whole genome sequencing of the isolates revealed missense mutations almost exclusively in TPR motifs 9 and 10 of the transcriptional co-repressor Cyc8 that specifically mediate repression of Mig1-targeted genes. The only other mutation occurred in MIG1 itself, clearly implicating the activation of Mig1-repressed genes as a key function of SNF1 in maintaining CLS. Consistent with this conclusion, the cyc8 TPR mutations partially restored growth on alternative carbon sources and significantly extended CLS compared to the snf1∆ parent. Furthermore, cyc8 TPR mutations also reactivated the Mig1-repressed transcription factor gene CAT8, which is responsible for activating genes of the glyoxylate and gluconeogenesis pathways. Deleting CAT8 completely blocked CLS extension by the cyc8 TPR mutations on CLS, identifying these pathways as key Snf1-regulated CLS determinants.