Emily O. Kerr

Regulation of Yeast Replicative Life Span by TOR and Sch9 in Response to Nutrients

Calorie restriction increases life span in many organisms, including the budding yeast Saccharomyces cerevisiae. From a large-scale analysis of 564 single-gene–deletion strains of yeast, we identified 10 gene deletions that increase replicative life span. Six of these correspond to genes encoding components of the nutrient-responsive TOR and Sch9 pathways. Calorie restriction of tor1D or sch9D cells failed to further increase life span and, like calorie restriction, deletion of either SCH9 or TOR1 increased life span independent of the Sir2 histone deacetylase. We propose that the TOR and Sch9 kinases define a primary conduit through which excess nutrient intake limits longevity in yeast.

Yeast Life Span Extension by Depletion of 60S Ribosomal Subunits Is Mediated by Gcn4

In nearly every organism studied, reduced caloric intake extends life span. In yeast, span extension from dietary restriction is thought to be mediated by the highly conserved, nutrient-responsive target of rapamycin (TOR), protein kinase A (PKA), and Sch9 kinases. These kinases coordinately regulate various cellular processes including stress responses, protein turnover, cell growth, and ribosome biogenesis. Here we show that a specific reduction of 60S ribosomal subunit levels slows aging in yeast. Deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span. One mechanism by which reduced 60S subunit levels leads to life span extension is through induction of Gcn4, a nutrient-responsive transcription factor. Genetic epistasis analyses suggest that dietary restriction, reduced 60S subunit abundance, and Gcn4 activation extend yeast life span by similar mechanisms.

Quantitative evidence for conserved longevity pathways between divergent eukaryotic species

Studies in invertebrate model organisms have been a driving force in aging research, leading to the identification of many genes that influence life span. Few of these genes have been examined in the context of mammalian aging, however, and it remains an open question as to whether and to what extent the pathways that modulate longevity are conserved across different eukaryotic species. Using a comparative functional genomics approach, we have performed the first quantitative analysis of the degree to which longevity genes are conserved between two highly divergent eukaryotic species, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Here, we report the replicative life span phenotypes for single-gene deletions of the yeast orthologs of worm aging genes. We find that 15% of these yeast deletions are long-lived. In contrast, only 3.4% of a random set of deletion mutants are long-lived-a statistically significant difference. These data suggest that genes that modulate aging have been conserved not only in sequence, but also in function, over a billion years of evolution. Among the longevity determining ortholog pairs, we note a substantial enrichment for genes involved in an evolutionarily conserved pathway linking nutrient sensing and protein translation. In addition, we have identified several conserved aging genes that may represent novel longevity pathways. Together, these findings indicate that the genetic component of life span determination is significantly conserved between divergent eukaryotic species, and suggest pathways that are likely to play a similar role in mammalian aging.

Integrative phenomics reveals insight into the structure of phenotypic diversity in budding yeast

To better understand the quantitative characteristics and structure of phenotypic diversity, we measured over 14,000 transcript, protein, metabolite, and morphological traits in 22 genetically diverse strains of Saccharomyces cerevisiae. More than 50% of all measured traits varied significantly across strains [false discovery rate (FDR) = 5%]. The structure of phenotypic correlations is complex, with 85% of all traits significantly correlated with at least one other phenotype (median = 6, maximum = 328). We show how high-dimensional molecular phenomics data sets can be leveraged to accurately predict phenotypic variation between strains, often with greater precision than afforded by DNA sequence information alone. These results provide new insights into the spectrum and structure of phenotypic diversity and the characteristics influencing the ability to accurately predict phenotypes.

The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function

The peroxisome, sole site of β-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified ∼130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity. The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190Δ, sit4Δ, and ilm1Δ strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids.

Sirtuin-independent effects of nicotinamide on lifespan extension from calorie restriction in yeast

Two models have been proposed for how calorie restriction (CR) enhances replicative longevity in yeast: (i) suppression of rDNA recombination through activation of the sirtuin protein deacetylase Sir2 or (ii) decreased activity of the nutrient‐responsive kinases Sch9 and TOR. We report here that CR increases lifespan independently of all Sir2‐family proteins in yeast. Furthermore, we demonstrate that nicotinamide, an inhibitor of Sir2‐mediated deacetylation, interferes with lifespan extension from CR, but does so independent of Sir2, Hst1, Hst2, and Hst4. We also find that 5 mm nicotinamide, a concentration sufficient to inhibit other sirtuins, does not phenocopy deletion of HST3. Thus, we propose that lifespan extension by CR is independent of sirtuins and that nicotinamide has sirtuin‐independent effects on lifespan extension by CR.

Design and use of multiplexed chemostat arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. We present here the design and operation of a relatively simple, low cost array of miniature chemostats—or ministats—and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

Chemostat Culture for Yeast Physiology

The use of chemostat culture facilitates the careful comparison of different yeast strains growing in well-defined conditions. Variations in physiology can be measured by examining gene expression, metabolite levels, protein content, and cell morphology. In this protocol, we show how a combination of sample types can be collected during harvest from a single 20-mL chemostat in a ministat array, with special attention to coordinating the handling of the most time-sensitive sample types.