Characterization of molecular mechanisms of aging in Saccharomyces cerevisiae

Dietary restriction (DR), which is the reduction of nutrients while avoiding malnutrition, extends lifespan and reduces age-associated pathologies of nearly every organism studied. In the yeast S. cerevisiae, replicative lifespan is defined as the number of daughter cells produced by one mother, and can be extended by up to 40% by DR. This lifespan extension is thought to be mediated by 3 kinases: TOR (target of rapamycin), PKA (protein kinase A) and Sch9. In response to nutrients, these highly conserved kinases coordinately regulate multiple processes central to cell biology, including autophagy, stress response pathways, cell growth and ribosome biogenesis. The myriad cellular processes regulated by these kinases make it difficult to elucidate the functions responsible for mediating lifespan extension. That orthologs of TOR, PKA and Sch9 have been shown to modulate aging in multicellular eukaryotes, including mice, warrants further investigation into the downstream cellular processes responsible for extending lifespan. With short lifespans and easily-manipulated genomes, S. cerevisiae serves as an ideal model organism for characterizing these molecular mechanisms. Toward this goal, we have employed a multi-faceted approach including both in-depth investigation of a specific cellular process, ribosome biogenesis, as well as large-scale genetic epistasis analyses.;A partial screen for genes whose deletion results in lifespan extension identified two ribosomal protein genes (RPGs), suggesting that perhaps ribosome biogenesis, which is regulated by TOR, PKA and Sch9, is a key determinant of lifespan. A targeted screen of all the RPG deletion strains revealed that deletion of many, but not all, RPGs extend lifespan in yeast. Strikingly, all the RPGs identified encode 60S subunit proteins. The data here demonstrate that reduced 60S ribosomal subunit abundance (by deletion of 60S RPGs, 60S subunit processing factor genes, or treatment with diazaborine) correlates with lifespan extension in yeast. One mechanism by which this occurs is through enhanced translation of GCN4, a highly conserved nutrient-responsive transcription factor.;In addition to influencing lifespan, ribosomal proteins (RPs) have been identified in a large number of genome-wide screens for various other phenotypes. However, a strong selection for suppressors of growth rate defects in these strains can complicate phenotypic observations. Therefore, we regenerated the entire set of haploid yeast RPG deletion strains, allowing us to define, for the first time, the set of fourteen nonessential RPs in yeast. This set of RPG deletion strains was then used to measure the response to tunicamycin treatment. A marked correlation between reduced growth rate and resistance to tunicamycin was observed, suggesting that reduced translation is protective against ER stress. The data here support a hypothesis that lifespan extension by reduced translation in multiple model organisms may, in part, be due to relief of ER stress.;Finally, in a broad approach to identifying molecular mechanisms of aging, we have performed large-scale genetic epistasis analysis to identify cellular processes required for lifespan extension by each of six highly conserved genetic modulators of aging. The general strategy for genetic epistasis analysis is to combine two mutants which affect aging and examine the phenotype of the double mutant. The interpretation of such data is often very complicated; therefore, we propose a systematic method for analyzing and interpreting genetic epistasis data in aging research. This method is then applied to a large-scale genetic epistasis analysis of yeast lifespan. The roles of at least nine downstream factors known to influence aging are examined, including autophagy, ribosome biogenesis and several stress response pathways. These data indicate that lifespan extension by different genetic modulators can require both overlapping and distinct cellular functions.;This work provides a foundation for understanding the cellular processes which modulate aging in yeast. The high level of conservation among the genetic modulators of aging investigated here merits future investigation of their roles in lifespan regulation in multicellular eukaryotes. Ultimately, these studies may illuminate targets for inhibition of age-associated pathologies.