Project description:The regulation of cell cycle progression in response to environmental cues is essential for cellular adaptation. In Saccharomyces cerevisiae, the BAR1 gene modulates sensitivity to the mating pheromone α-factor, which induces cell cycle arrest. Here we investigated the dynamic proteomic response in the bar1 deletion strain using a 27-plex experimental design with TMTproD isobaric labeling. Asynchronous bar1Δ cells were arrested with α-factor and then released from the pheromone arrest. We acquired three replicate protein abundance time-course profiles following pheromone (α-factor) washout, with samples collected at eight time points from 0 to 165 minutes post-washout. Using higher-order TMTpro sample multiplexing, we generated global temporal profiles of protein abundance associated with recovery from pheromone-induced arrest. Our findings identify specific proteins and pathways involved in cell cycle re-entry and in the attenuation of the pheromone signal, providing insights into the regulatory mechanisms of mating response in yeast. This study contributes significantly to dynamic proteomic analysis in cell cycle progression. We present a powerful approach for investigating complex cellular processes and showcase cell cycle progression following pheromone washout in yeast.
Project description:We measured the response of S. cerevisiae to arrest in the presence of alpha factor. These were collected in support of a related DNaseI-sequencing study. Keywords: Alpha-factor arrest S.cerevisiae R276 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 bar1Δ::KanMX) (C. Boone, University of Toronto; S288c background derived from BY4741), was cultured overnight in 50 ml rich medium (YPD) at 30°C, diluted into 500 ml fresh YPD to an OD660 of ~0.8, and treated with yeast α-factor (Sigma-Aldrich) at a final concentration of 50 ng / ml. This culture was incubated at 30°C with shaking for 3 hours (final OD660 ~1). After this treatment, approximately 90% of the cells had formed mating projections when checked by light microscopy. Total RNA from these cells was isolated using hot acidic phenol. 50 μg of total RNA was treated with Turbo Dnase (Ambion), and checked for integrity using a Bioanalyzer 2100 (Agilent). Total RNA was labeled according to the manufacturer’s protocol and applied to Affymetrix Yeast 2.0 arrays. Data were analyzed using the “affy” package from Bioconductor.
Project description:Transcript profiling from a population of cell-cycle-arrested B-type cyclin mutant budding yeast. We compared transcript to protein expression levels in mutant cells.