Layers of regulation on cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae
Kelliher CM, Foster MW, Motta FC, Deckard A, Soderblom EJ, Moseley MA, Haase SB. Layers of regulation on cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae. Mol Biol Cell [Internet]. 2018 Sep 12;mbc.E18-04-0255.
- Organism: Saccharomyces cerevisiae
- Instrument: Q Exactive Plus
yeast, transcription factor, time-course
In the budding yeast Saccharomyces cerevisiae, transcription factors (TFs) regulate the periodic expression of many genes during the cell cycle, including gene products required for progression through cell-cycle events. Experimental evidence coupled with quantitative models suggest that a network of interconnected TFs is capable of regulating periodic genes over the cell cycle. Importantly, these dynamical models were built on transcriptomics data and assumed that TF protein levels and activity are directly correlated with mRNA abundance. To ask whether TF transcripts match protein expression levels as cells progress through the cell cycle, we applied a multiplexed targeted mass spectrometry approach (parallel reaction monitoring) on synchronized populations of cells. We found that protein expression of many TFs and cell-cycle regulators closely followed their respective mRNA transcript dynamics in cycling wild-type cells. Discordant mRNA/protein expression dynamics were also observed for a subset of cell-cycle TFs and for proteins targeted for degradation by E3 ubiquitin ligase complexes such as SCF (Skp1/Cul1/F-box) and APC/C (anaphase-promoting complex/cyclosome). We further profiled mutant cells lacking B-type cyclin/CDK activity (clb1-6), where oscillations in ubiquitin ligase activity, cyclin/CDKs, and cell-cycle progression are halted. We found that a number of proteins were no longer periodically degraded in clb1-6 mutants compared to wild type, highlighting the importance of post-transcriptional regulation. Finally, the TF complexes responsible for activating G1/S transcription (SBF and MBF) were more constitutively expressed at the protein level than their periodic mRNA expression levels in both wild-type and mutant cells. This comprehensive investigation of cell-cycle regulators reveals that multiple layers of regulation (transcription, protein stability, and proteasome targeting) affect protein expression dynamics during the cell cycle.
A PRM assay was developed to target 45 yeast cell-cycle proteins and 4 constitutively-expressed controls. Following reduction/alkylation and tryptic digestion of yeast lysates, samples were spiked with stable isotope-labeled peptides at 5 fmol SIL per ug proteins. 1 µg of peptide digests per sample were analyzed using a nanoACQUITY UPLC (Waters) and QExactive Plus MS (Thermo). Time course samples were analyzed in a random order and interspersed with QC pool samples containing equal mixtures of all samples. Data collection on the QExactive Plus MS was performed in targeted MS/MS mode at 17,500 resolution (m/z 200) with a target AGC value of 5e4 ions, an isolation width of 1.0 m/z and an ion fill time of 240 ms. Targeted MS/MS was triggered by an inclusion list, with 2 minute retention time windows for each precursor.
Saccharomyces cerevisiae strains are derivatives of BF264-15D MATa bar1. Wild-type or PGAL1-CLB1 clb1-6Δ cells were synchronized using alpha factor and released into YEPD. Wild-type cells were collected on filters every 7 minutes across 20 time points (2 replicates). clb1-6Δ cells were collected on filters every 7 minutes across 21 time points (2 replicates)
Created on 8/30/18 9:58 AM