Comprehensive Temporal Protein Dynamics during the Diauxic Shift in Saccharomyces cerevisiae

Abstract

Yeast (Saccharomyces cerevisiae) has served as a key model system in biology and as a benchmark for "omics" technology. Although near-complete proteomes of log phase yeast have been measured, protein abundance in yeast is dynamic, particularly during the transition from log to stationary phase. Defining the dynamics of proteomic changes during this transition, termed the diauxic shift, is important to understand the basic biology of proliferative versus quiescent cells. Here, we perform temporal quantitative proteomics to fully capture protein induction and repression during the diauxic shift. Accurate and sensitive quantitation at a high temporal resolution and depth of proteome coverage was achieved using TMT10 reagents and LC-MS3 analysis on an Orbitrap Fusion tribrid mass spectrometer deploying synchronous precursor selection. Triplicate experiments were analyzed using the time-course R package and a simple template matching strategy was used to reveal groups of proteins with similar temporal patterns of protein induction and repression. Within these groups are functionally distinct types of proteins such as those of glyoxylate metabolism and many proteins of unknown function not previously associated with the diauxic shift (e.g. YNR034W-A and FMP16). We also perform a dual time-course experiment to determine Hap2-dependent proteins during the diauxic shift. These data serve as an important basic model for fermentative versus respiratory growth of yeast and other eukaryotes and are a benchmark for temporal quantitative proteomics.

Fig. 1.

Comprehensive and temporal proteomic profiling of the diauxic shift in yeast using TMT10. A, Proteomic sampling and labeling scheme. In triplicate, 10 culture aliquots were acquired over a 33 h time period following inoculation, spanning the logarithmic and stationary phases of growth. Cells were lysed, digested, labeled with TMT10 reagents and combined to a single sample per replicate. Each sample was then separated into 12 fractions by basic-pH reversed phase chromatography and each fraction was analyzed by LC-MS3 using an Orbitrap Fusion mass spectrometer. B, Glucose and OD_{600 nm} throughout the course of the experiment. C, Heatmap of relative intensities (% of the total TMT reporter ion signal) for each protein representing abundance changes for 4547 proteins across 10 time-points during the diauxic shift. D, Specific examples of temporal protein profile data and validation using immunoblotting of TAP-tagged strains grown to log (L) or stationary phase (S). Error bars represent the standard error of triplicate protein relative intensity measurements.

Fig. 2.

Statistical analysis of the triplicate Fusion data sets. All proteins measured in at least two of the replicate analyses were assigned a T² statistic using the time-course package in R Bioconductor. Shown are the calculated log₁₀ T²-statistics plotted against the log₂ values for the 33h/5h time point ratios. Examples are shown demonstrating the utility of the raw time-course-generated T²-statistics in assessing temporal changing proteins.

Fig. 3.

Time-course application of TMT10 facilitates comprehensive temporal protein profiling, differentiating between early and late changes in protein abundance during the diauxic shift. A, Proteins from the data set were assessed for significance using the time-course package in R, then temporally moderated proteins were matched to specific template protein profiles as shown; early-induced (899 proteins total), late-induced (69 proteins total), induced then repressed (10 proteins total), early-repressed (1040 proteins total), or late-repressed (72 proteins). Matching was based on Euclidean distance between 10-plex TMT protein profiles and the given template. Over-represented GO terms were assigned to the list of proteins matching each template profile using DAVID with a Benjamini Hochberg corrected p value < 0.05. Examples of proteins from over-represented GO terms (if assigned) are shown for B, late-induced, C, early-induced, D, induced then repressed, E, early-repressed, and F, late-repressed proteins.

Fig. 4.

Correlation of the time-course TMT10 data set with mRNA data from Derisi et al. A, Aligned heatmaps of time-course profiles between the two studies. To directly compare the two studies, data are expressed as log₂ ratios to the earliest time point (proteins shown appear in all 3 replicate TMT10 analyses). B, The latest time-points are compared with the earliest time-points for the log₂ ratios in both studies. Genes or proteins in the top-left and bottom-right quadrants differ between this study and Derisi et al. C, Examples of several proteins induced or repressed in this study (at the protein level) but not in the data set of Derisi et al. (mRNA level).

Fig. 5.

Differences in the induction of specific isoforms of various metabolically important proteins during the diauxic shift; A, Aldehyde dehydrogenases, B, Alcohol dehydrogenases, C, Glyceraldehyde-3-phosphate dehydrogenases, and D, Glutamate dehydrogenases. Error bars represent the standard error of triplicate protein relative intensity measurements.

Fig. 6.

Dual time-course TMT10 profiling of Hap2-regulated proteins during the diauxic shift. A, Proteomic sampling and labeling scheme. Five aliquots from BY4742 WT and ΔHap2 strains were acquired over a 24 h time period following inoculation, spanning the logarithmic and stationary phases of growth. Cells were lysed, digested, labeled with 10-plex reagents and combined to a single sample. The sample was then separated into 12 fractions by basic-pH reversed phase chromatography and each fraction was analyzed by LC-MS3. B, Reproducibility between experiments represented by aligned heatmaps of relative intensities (% of the total for each protein) for the initial 10-plex time-course and the 10-plex dual time-course. C, Proteins with relative intensity differences between WT (Blue) and ΔHap2 (Red) during the diauxic shift. D, Validation of PCK1, ACH1, and AGP2 dependence on Hap2. Each protein was TAP-tagged in the ΔHap2 strain and measured by immunoblot in a separate experiment in either log (L) or stationary (S) phase.