Proteome analysis of yeast response to various nutrient limitations

Abstract

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose‐repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β‐oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post‐transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology. ### Synopsis Organisms, varying from unicellular bacteria to mammalian species, rely on the uptake of nutrients from the environment to sustain energy, metabolism and growth. They have therefore evolved numerous strategies to adapt to their changing environment. Such programs involve instantaneous responses (changes in intracellular metabolites, activation/inhibition of enzymes by effectors and of proteins through post‐translational modifications) as well as slower processes that affect the levels of macromolecules (transcription, translation, mRNA and protein degradation). The yeast Saccharomyces cerevisiae can adapt very well to different environments, and is therefore an ideal system to investigate the molecular system response to such changes in the environment. In recent large‐scale transcriptome analyses of S. cerevisiae grown under various nutrient limitations ([Boer et al , 2003][1]; [Daran‐Lapujade et al , 2004][2]; [Saldanha et al , 2004][3]; [Wu et al , 2004b][4]; [Tai et al , 2005][5]), specific transcriptional responses to the limiting nutrient were identified. However, it is not known to what extent these major transcriptional responses are actually translated into quantitatively identical responses at the protein level. With the aim of investigating the proteomic response of S. cerevisiae grown under different nutrient limitation regimes (carbon and nitrogen limitation) and to assess to what extent changes at the protein level are reflected in changes at the transcriptional level, we performed an extensive quantitative proteome analysis of chemostat‐cultivated S. cerevisiae limited for glucose or ammonia ([Figure 1][6]). The yeast strains were therefore labeled in vivo by metabolic stable isotope labeling with 15N, enabling the differential quantification of protein expression levels directly by mass spectrometry ([Ong et al , 2003][7]; [Romijn et al , 2003][8]). The experimental approach that combined isotope labeling with one‐dimensional gel separation of proteins and LC‐MS/MS analysis of peptides generated by tryptic proteolysis of 1D gel bands led to a quantitative data set that appeared to be less biased compared to standard 2D gel‐based analysis, as proteins from different subcellular compartments, including membrane proteins, and proteins with extreme isoelectric points and molecular weights were identified. In order to reduce error and noise in the data, we used rather stringent criteria to filter the protein expression levels, resulting in a data set of 102 proteins that were considered as significantly changed. We also performed a reverse isotope labeling experiment from which we concluded that the reproducibility of the expression ratios was within 20% at least for 3/4 of the quantified proteins, reflecting a good experimental and biological reproducibility. Additionally, we performed multiple LC‐MS/MS analysis, and analyzed our data stringently, leading to the quantification of about 750 proteins, with an average of nearly five peptide pairs per protein. We believe that this is at present one of the best quality quantitative proteomics studies, enabling a proper comparison with transcriptome data. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose‐repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β‐oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging alternative nitrogen sources and in protein degradation. Our data set also revealed numerous proteins that seem to be present rather exclusively under either nitrogen or carbon limitation. Some of these proteins had poorly described functions, but are likely important for survival under carbon or nitrogen limitation. We also examined whether proteins known to be components of functional and/or structural protein complexes showed differential coexpression. Interestingly, we observed that there is a good correlation in coexpression for various protein complexes, such as the vacuolar H+‐ATPase complex, the cytochrome c oxidase complex and the proteasome. However, for some complexes (e.g. the F0/F1 ATP synthase), opposite regulation of the different subunits was observed, making overall conclusions not straightforward. Our extensive and high‐quality data set enabled us to compare our...