Patrick A. Gibney

Yeast metabolic and signaling genes are required for heat-shock survival and have little overlap with the heat-induced genes

Significance We used the model eukaryote Saccharomyces cerevisiae to investigate which genes are important for survival of heat stress. Previously, this question was addressed by examining which genes are turned on by mild heat stress; in this study, we examined gene-deletion mutants for increased sensitivity to lethal heat stress. This approach reveals that these two sets of genes are largely nonoverlapping, demonstrating that mutant analysis is a powerful complementary approach to gene-expression analysis. In addition, many of the genes identified as important for heat survival are involved in metabolism or signaling, or their function is completely uncharacterized, suggesting that our understanding of the systems-level response to heat stress is incomplete. Genome-wide gene-expression studies have shown that hundreds of yeast genes are induced or repressed transiently by changes in temperature; many are annotated to stress response on this basis. To obtain a genome-scale assessment of which genes are functionally important for innate and/or acquired thermotolerance, we combined the use of a barcoded pool of ∼4,800 nonessential, prototrophic Saccharomyces cerevisiae deletion strains with Illumina-based deep-sequencing technology. As reported in other recent studies that have used deletion mutants to study stress responses, we observed that gene deletions resulting in the highest thermosensitivity generally are not the same as those transcriptionally induced in response to heat stress. Functional analysis of identified genes revealed that metabolism, cellular signaling, and chromatin regulation play roles in regulating thermotolerance and in acquired thermotolerance. However, for most of the genes identified, the molecular mechanism behind this action remains unclear. In fact, a large fraction of identified genes are annotated as having unknown functions, further underscoring our incomplete understanding of the response to heat shock. We suggest that survival after heat shock depends on a small number of genes that function in assessing the metabolic health of the cell and/or regulate its growth in a changing environment.

Systems-level analysis of mechanisms regulating yeast metabolic flux

Quantitation of metabolic pathway regulation Although metabolic biochemical pathways are well understood, less is known about precisely how reaction rates or fluxes through the various enzymes are controlled. Hackett et al. developed a method to quantitate such regulatory influence in yeast. They monitored concentrations of metabolites, enzymes, and potential regulators by LC-MS/MS (liquid chromatography–tandem mass spectrometry) and isotope ratio measurements for 56 reactions, over 100 metabolites, and 370 metabolic enzymes in yeast in 25 different steady-state conditions. Bayesian analysis was used to examine the probability of regulatory interactions. Regulation of flux through the pathways was predominantly controlled by changes in the concentration of small-molecule metabolites rather than changes in enzyme abundance. The analysis also revealed previously unrecognized regulation between pathways. Science, this issue p. 432 Metabolomics, proteomics, and flux analysis are used to dissect quantitatively metabolic regulation in living yeast. INTRODUCTION Metabolism is among the most strongly conserved processes across all domains of life and is crucial for both bioengineering and disease research, yet we still have an unclear understanding of how metabolic rates (fluxes) are determined. Qualitatively, this deficiency involves missing knowledge of enzyme regulators. Quantitatively, it involves limited understanding of the relative contributions of enzyme and metabolite concentrations in controlling flux across physiological conditions. Addressing these gaps has been challenging because in vitro biochemical approaches lack the physiological context, whereas models of cellular metabolic dynamics have limited capacity for identifying or quantitating specific regulatory events because of overall model complexity. RATIONALE Flux through individual metabolic reactions is directly determined by the concentrations of enzyme, substrates, products, and any allosteric regulators, as captured quantitatively by a Michaelis-Menten–style reaction equation. Analogous to how experimental variation of reaction species in vitro allows for the inference of regulators and reaction equation kinetic parameters, physiological changes in flux entail a change in reaction species that can be used to determine reaction equations on the basis of cellular data. This requires measurement across multiple biological conditions of flux, enzyme concentrations, and metabolite concentrations. We reasoned that chemostat cultures could be used to induce predictable and strong flux changes, with changes in enzymes and metabolites measurable by proteomics and metabolomics. By directly relating cellular flux to the reaction species that determine it, we can carry out regulatory inference at the level of single metabolic reactions by using cellular data. An important benefit is that the physiological significance of any identified regulator is implicit from its role in determining cellular flux. RESULTS Here we introduce systematic identification of meaningful metabolic enzyme regulation (SIMMER). We measured fluxes, and metabolite and enzyme concentrations, in each of 25 yeast chemostats. For each of 56 reactions for which the flux, enzyme, and substrates were measured, we determined whether variation in measured flux could be explained by simple Michaelis-Menten kinetics. We also evaluated alternative models of each reaction’s kinetics that included a suite of allosteric regulators drawn from across all organisms. For 46 reactions, we were able to identify a useful kinetic model, with 17 reactions not including any regulation and 29 reactions being regulated by one to two allosteric regulators. Three previously unrecognized cross-pathway regulatory interactions were validated biochemically. These included inhibition of pyruvate kinase by citrate and inhibition of pyruvate decarboxylase by phenypyruvate. These metabolites accumulated and thereby curtailed glycolytic outflow and ethanol production in nitrogen-limited yeast. For well-supported reaction forms, we were able to determine the extent to which nutrient-based changes in flux were determined by changes in the concentrations of individual reaction species. We find that substrates are the most important determinant of fluxes in general, with enzymes and allosteric regulators having a comparably important role in the case of physiologically irreversible reactions. CONCLUSION By connecting changes in flux to their root cause, SIMMER parallels classic in vitro approaches, but it allows simultaneous testing of numerous regulators of many reactions under physiological conditions. Its application to yeast showed that changes in flux across nutrient conditions are predominantly due to metabolite, not enzyme, levels. Thus, yeast metabolism is substantially self-regulating. Integrative analysis of fluxes and metabolite and enzyme concentrations by SIMMER. Measured flux is related, on a reaction-by-reaction basis, to enzyme and metabolite concentrations through a Michaelis-Menten equation. The extent to which variation in flux across experimental conditions can be explained by the enzyme, substrates, and products is assessed. If unregulated kinetics disagrees with the measured flux, we test a set of possible allosteric regulators to determine which, if any, regulators are supported on the basis of improvement in fit. Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states and, therefore, how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three previously unrecognized instances of cross-pathway regulation, which we biochemically verified. One of these involved inhibition of pyruvate kinase by citrate, which accumulated and thereby curtailed glycolytic outflow in nitrogen-limited yeast. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes.

Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae

We developed systems to rapidly express any yeast gene or to specifically degrade any protein, each with minimal untargeted disturbance of cell physiology. We illustrate applications of these new tools for elucidating the architecture and dynamics of genetic regulatory networks.

Characterizing the in vivo role of trehalose in Saccharomyces cerevisiae using the AGT1 transporter

Significance Trehalose is an important molecule for industrial and medical applications. These applications include use as a food additive to increase sweetness and promote freeze-dry preservation. Trehalose is also included in antibody preparations for stabilization during freezing or desiccation. Further, trehalose biosynthesis is required for virulence of fungal pathogens, and, because animal cells do not synthesize trehalose, trehalose biosynthesis is an attractive antifungal target. Despite all of its uses, the direct physiological roles of trehalose remain unclear. Here, we describe the development and characterization of a system in the model yeast Saccharomyces cerevisiae to directly assess the physiological roles of trehalose. We find that many of the roles traditionally ascribed to trehalose are not the result of trehalose accumulation per se. Trehalose is a highly stable, nonreducing disaccharide of glucose. A large body of research exists implicating trehalose in a variety of cellular phenomena, notably response to stresses of various kinds. However, in very few cases has the role of trehalose been examined directly in vivo. Here, we describe the development and characterization of a system in Saccharomyces cerevisiae that allows us to manipulate intracellular trehalose concentrations independently of the biosynthetic enzymes and independently of any applied stress. We found that many physiological roles heretofore ascribed to intracellular trehalose, including heat resistance, are not due to the presence of trehalose per se. We also found that many of the metabolic and growth defects associated with mutations in the trehalose biosynthesis pathway are not abolished by providing abundant intracellular trehalose. Instead, we made the observation that intracellular accumulation of trehalose or maltose (another disaccharide of glucose) is growth-inhibitory in a carbon source-specific manner. We conclude that the physiological role of the trehalose pathway is fundamentally metabolic: i.e., more complex than simply the consequence of increased concentrations of the sugar and its attendant physical properties (with the exception of the companion paper where Tapia et al. [Tapia H, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1506415112] demonstrate a direct role for trehalose in protecting cells against desiccation).

Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae.

A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.

TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

Desiccation is thought to impose many stresses. Which of these stresses is responsible for desiccation-induced death and how the stress response is regulated are unknown, however. Here we use Saccharomyces cerevisiae to show that reduction of a 60S biogenesis intermediate via RAS or TOR down-regulation increases desiccation tolerance.

From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span

Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long‐lived naked mole‐rat, suggesting that it naturally presents with a life‐extending phenotype akin to that observed in methionine‐restricted animals. Similarly, long‐lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine‐restriction effects on life‐span extension, however, remain unknown, as do their potential connections with caloric restriction, another well‐established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span—from yeast to humans—and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span.

The Miniature-chemostat Aging Device: A new experimental platform facilitates assessment of the transcriptional and chromatin landscapes of replicatively aging yeast

Replicative aging of Saccharomyces cerevisiae is an established model system for eukaryotic cellular aging. A major limitation in yeast lifespan studies has been the difficulty of separating old cells from young cells in large quantities for in-depth comparative analyses. We engineered a new platform, the Miniature-chemostat Aging Device (MAD), that enables purification of aged cells at sufficient quantities to enable genomic and biochemical characterization of aging yeast populations. Using the MAD platform, we measured DNA accessibility (ATAC-Seq) and gene expression (RNA-Seq) changes in aging cells. Our data highlight an intimate connection between aging, growth rate, and stress, as many (but not all) genes that change with age have altered expression in cells that are subjected to stress. Stress-independent genes that change with age are highly enriched for targets of the signal recognition particle (SRP). By obtaining pure populations of old cells, we find that nucleosome occupancy does not change significantly with age; however, significant age-dependent changes in accessibility at ~12% of genomic loci reflect decreased replication and changing activities of cell cycle and metabolic regulators. Finally, ATAC-seq revealed that upregulating the proteasome by deleting UBR2 reduces rDNA instability usually observed in aging cells, demonstrating a connection between proteasome activity and genomic stability.

Minor isozymes tailor yeast metabolism to carbon availability

Isozymes are enzymes that differ in sequence but catalyze the same chemical reactions. Despite their apparent redundancy, isozymes are often retained over evolutionary time for reasons that can be unclear. We find that, in yeast, isozymes are strongly enriched in central carbon metabolism. Using a gene expression compendium, we find that many isozyme pairs show anticorrelated expression during the respirofermentative shift, suggesting roles in adapting to changing carbon availability. Building on this observation, we assign function to two minor central carbon isozymes, aconitase 2 (ACO2) and pyruvate kinase 2 (PYK2). ACO2 is expressed during fermentation and proves advantageous when glucose is limiting. PYK2 is expressed during respiration and proves advantageous for growth on three-carbon substrates. PYK2’s deletion is rescued by expressing the major pyruvate kinase, but only if that enzyme carries mutations mirroring PYK2’s allosteric regulation. Thus, central carbon isozymes enable more precise tailoring of metabolism to changing nutrient availability. Importance Gene duplication is one of the main evolutionary drivers of new protein function. However, some gene duplicates have nevertheless persisted long-term without apparent divergence in biochemical function. Further, under standard lab conditions, many isozymes have subtle or no knockout phenotypes. These factors make it hard to assess the unique contributions of individual isozymes to fitness. We therefore developed a method to identify experimental perturbations that could expose such contributions, and applied it to yeast gene expression data, revealing a potential role for a set of yeast isozymes in adapting to changing carbon sources. Our experimental confirmation of distinct roles for two “minor” yeast isozymes, including one with no previously described knockout phenotype, highlight that even apparently redundant paralogs can have important and unique functions, with implications for genome-scale metabolic modeling and systems-level studies of quantitative genetics.

Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.