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Quantitative proteomics of anaerobic and aerobic yeast cultures

ABSTRACT: Saccharomyces cerevisiae is unique among yeasts for its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of the S. cerevisiae to anaerobiosis was investigated using metabolic stable isotope labeling in aerobic and anaerobic glucose-limited chemostat cultures, followed by proteome analysis to relatively quantify protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many responses of S. cerevisiae to oxygen availability were hitherto unreported. Comparison of transcriptome and proteome from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl tRNA synthesis, purine-nucleotide synthesis and amino-acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights in the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the messenger RNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology. Protein levels versus transcript level: Systematic analysis of the control levels at which the yeast response to anaerobiosis takes place was performed using previously published transcript data obtained from yeast cultures grown under strictly identical conditions as described for the current proteome analysis. Affymetrix microarrays from five aerobic and four anaerobic independent culture replicates were used for this analysis. These comparison data are summarized in the table below. These array data are publicly available at the gene expression repository Gene Expression Omnibus under accession number GSE4804. Keywords: proteomic, nanoflow-LC-MS/MS

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE7365 | GEO | 2007/09/26

SECONDARY ACCESSION(S): PRJNA100439

REPOSITORIES: GEO

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Project description:Despite the scientific and applied interest in anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is up-regulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically up-regulated TIR1 gene was fused to LacZ revealed a complete loss of anaerobic up-regulation in a snf7Δ mutant. Anaerobic up-regulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p transcription factor. Analysis of LacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic up-regulation of TIR1 involved Rim101p and did not require a functional multi-vesicular body sorting pathway (in which Snf7p also participates). Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited a Snf7p/Rim101p dependent anaerobic up-regulation. Since, in its activated form, Rim101p is generally known as a transcriptional repressor, its role in anaerobic up regulation of TIR1 and other ‘anaerobic’ yeast genes must involve additional factors. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, showed that these genes were not involved in the regulation of TIR1. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p complex. The physiological relevance of Snf7p/Rim101p-mediated transcriptional up-regulation of several genes in anaerobic yeast cultures was evident from reduced growth of a snf7Δ under anaerobic conditions. Experiment Overall Design: The aim of the present study is to investigate the role of SNF7 in the regulation of TIR1, to assess whether this role involves the Rim101p pathway, and to investigate whether Snf7p and/or Rim101p are also involved in regulation of other ‘anaerobic’ S. cerevisiae genes. After analyzing transcriptional regulation of TIR1 in several genetic backgrounds, a chemostat-based transcriptome analysis was performed on snf7Δ and rim101Δ strains as well as on the isogenic reference strain. Sets of genes that were differentially expressed in the snf7 and rim101 deletion strains were then compared to sets of genes that were previously shown to be transcriptionally up-regulated in anaerobic chemostat cultures of S. cerevisiae (Piper et al., 2002; Tai et al., 2005) Experiment Overall Design: Piper MD, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008. Experiment Overall Design: Tai SL, Boer VM, Daran-Lapujade P, Walsh MC, de Winde JH, Daran JM & Pronk JT (2005) Two-dimensional transcriptome analysis in chemostat cultures. Combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae. J Biol Chem 280: 437-447.

Project description:Saccharomyces cerevisiae is currently widely used as a model to study chronological aging of metazoan cells. Chronological aging is typically studied in aerobic stationary phase (SP) cultures, i.e. the final stage of batch cultures in which growth is arrested due to exogenous carbon source exhaustion. Survival of yeast cells in SP defines their chronological lifespan (CLS). S. cerevisiae SP cultures have strongly contributed to the understanding of cellular mechanisms involved in aging and indicated a key role for oxygen. Oxygen is the natural starting point for reactive oxygen species (ROS) that may both have malignant and beneficial effects on aging. In addition, oxygen allows yeast to grow on ethanol and organic acids formed during the initial respiro-fermentative growth phase on glucose. This post-diauxic phase is hallmarked by reduced growth rates, increased expression of genes involved in SP survival, and increased stress resistance. To date, the role of oxygen and respiration in aging has mostly been studied using respiratory deficient mutants, and respiration repressing agents. However, genetic or chemical interventions may result in unwanted side effects that influence survival in SP. We therefore followed a different approach to evaluate the impact of oxygen availability on yeast robustness in SP, i.e. its CLS and stress resistance, by using the capability of S. cerevisiae to grow under anaerobic conditions. A thorough physiological comparison of strictly anaerobic and aerobic SP cultures revealed that the presence of oxygen during growth and aging of S. cerevisiae strongly affects its energetic status, longevity and stress tolerance in a positive way. Combining the physiological data with genome-wide expression analysis revealed that the oxygen-dependent diauxic growth phase enabled the full induction of robustness in S. cerevisiae, and points to appropriate pre-conditioning of cells as a crucial factor to survive starvation. These findings highlight the importance of exogenous energy availability in the conditions leading to growth arrest, and bring new insight on the role of oxygen in the aging of eukaryotes.

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Quantitative proteomics of anaerobic and aerobic yeast cultures

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