Abstract
Chronologically aging yeast cells are prone to adaptive regrowth, whereby mutants with a survival advantage spontaneously appear and re-enter the cell cycle in stationary phase cultures. 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 in chronologically aging cells, including glucose depletion and oxidative stress. SNF1 is also required for the extension of chronological lifespan (CLS) by caloric restriction (CR) as defined as limiting glucose at the time of culture inoculation. To identify specific downstream SNF1 targets responsible for CLS extension during CR, we screened for adaptive regrowth mutants that restore chronological longevity to a short-lived snf1∆ parental strain. Whole genome sequencing of the adapted mutants revealed missense mutations in TPR motifs 9 and 10 of the transcriptional co-repressor Cyc8 that specifically mediate repression through the transcriptional repressor Mig1. Another mutation occurred in MIG1 itself, thus 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 reactivated multiple Mig1-repressed genes, including the 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.
Highlights
The budding yeast SNF1 complex is homologous to metazoan AMP-activated protein kinase (AMPK), and acts as a sensor of cellular energy status that adjusts metabolism in response to environmental nutrient conditions and stress [1,2,3]
Since SNF1 targets multiple transcription factors, metabolic enzymes, and even histone H3 [27], we hypothesized that mutations in genes allowing snf1∆ cells to survive in stationary phase during chronological aging would reveal critical targets or cellular processes that Snf1 regulates to promote chronological lifespan (CLS)
Suppressor mutations were originally identified in snf1 mutant strains by isolating revertant colonies that grew on sucrose-containing or other types of alternative carbon sources [38, 39]
Summary
The budding yeast SNF1 complex is homologous to metazoan AMP-activated protein kinase (AMPK), and acts as a sensor of cellular energy status that adjusts metabolism in response to environmental nutrient conditions and stress [1,2,3]. Snf phosphorylation of Acc inactivates the enzyme, promoting fatty acid catabolism and accumulation of acetyl CoA, which contributes to histone acetylation and transcriptional activity stimulated by low glucose [9]. Snf functions more directly in transcriptional activation by phosphorylating several transcription factors (Mig, Cat and others) that are responsible for diminishing glycolysis/fermentation pathways and promoting the utilization of alternative carbon sources such as ethanol and acetate [2]. This transition in transcription and metabolism is known as the diauxic shift, and is important for long-term cell survival in stationary phase cultures [10]
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