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 beta-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.

Figure 1

Schematic overview of quantitative proteomics approach used to study the effect of nutrient limitation on the yeast proteome.

Figure 2

Comparison of the properties of the 928 identified proteins and the total S. cerevisiae proteome. (A) Virtual 2D gel of the identified proteins (black dots) and of all ORFs of S. cerevisiae (gray dots). The area covered by a typical 2D gel (pI 4–9, Mw 15–150 kDa) is indicated with a dashed rectangle. (B) Distribution of the CAI of the proteins identified in this study and of the complete yeast proteome. (C) Subcellular localization distribution of the 928 identified proteins (left) and of all ORFs of S. cerevisiae (right) as defined by the Saccharomyces Genome Database. The percentage of proteins present in each subcellular localization is given in parentheses.

Figure 3

Typical examples of extremely regulated proteins. (A) The panel on the left side shows the extracted ion chromatograms of the ¹⁴N- and ¹⁵N-labeled version of the DIDIEYHQNK peptide from Hsp26p, which could only be detected in the ion chromatogram of the carbon-limited culture (¹⁴N version). The panel on the right shows the extracted ion chromatograms of the ¹⁴N- and ¹⁵N-labeled GTMITLNDR peptide from Asp3p. This peptide is only present in the nitrogen-limited yeast culture (¹⁵N version). (B) Extracted ion chromatograms from the same peptides as in panel A are shown for the reverse labeling experiment. In this experiment, the nitrogen-limited culture was grown on ¹⁴N media and the carbon-limited culture on ¹⁵N-labeled media.

Figure 4

Scatter plot of labeling and reverse labeling experiments. Only proteins that were quantified on the basis of at least three peptide peak pairs in both experiments were considered in this comparison (n=156). The solid line in the graph indicates a perfect correlation between the two experiments. The dashed lines indicate a 20% deviation in protein ratios.

Figure 5

Protein ratio distribution and the number of peptide pairs used for relative quantification. (A) Distribution of the 759 proteins as a function of the number of peptide pairs used for quantification. (B) Plot of relative protein expression levels from the carbon-limited versus the nitrogen-limited chemostat culture of all 759 relatively quantified proteins.

Figure 6

Sorting of the differentially expressed proteins as a function of the specific response to either glucose excess or ammonia limitation. The asterisk highlights a matching change in transcript level (C/N ratio greater than 1.5 or smaller than 0.66 and P-value below 0.05) (Boer et al, 2003). Arrows indicate a positive regulation, whereas bars indicate negative control.

Figure 7

Scatter plot of protein ratios versus mRNA expression ratios between carbon- and nitrogen-limited chemostats. The mRNA expression ratios were measured using oligonucleotide microarrays from rigorously identical culture conditions and were previously published by Boer et al (2003). The plotted x=y line indicates data points showing perfect correlation between mRNA and proteins abundances. Dashed lines indicate a two-fold deviation between mRNA and protein expression ratios. (A) All proteins quantified on the basis of five peptide pairs or more (n=278) were plotted. (B) Proteins that are differentially expressed are plotted (see Tables IIA and B). As ‘on/off' proteins were excluded from statistical analysis, only the remaining 26 proteins upregulated under glucose limitation and 40 proteins upregulated under ammonia limitation were plotted.

Figure 8

Changes in expression of transcripts and proteins (carbon versus nitrogen limitation) involved in fatty acids β-Oxidation (A) and ammonia incorporation into organic nitrogen (B). Proteins quantified in this study with their corresponding microarray mRNA level (left box) and protein level (right box) are indicated in bold.