Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program
Dissertations, UMMS; G1 Phase Cell Cycle Checkpoints; Pheromones; Saccharomyces cerevisiae
Cell Biology | Cellular and Molecular Physiology | Molecular Biology
Progression through the cell cycle is tightly controlled, and the decision whether or not to enter a new cell cycle can be influenced by both internal and external cues. For budding yeast one such external cue is pheromone treatment, which can induce G1 arrest. Two distinct mechanisms are known to be involved in this arrest, one dependent on the arrest protein Far1 and one independent of Far1, but the exact mechanisms have remained enigmatic. The studies presented here further elucidate both of these mechanisms.
We looked at two distinct aspects of the Far1-independent arrest mechanism. First, we studied the role of the G1/S regulatory system in G1 arrest. We found that deletion of the G1/S transcriptional repressors Whi5 and Stb1 compromises Far1-independent arrest, but only partially, and that this partial arrest failure correlates to partial de-repression of G1/S transcripts. Deletion of the CKI Sic1, however, is more strongly required for arrest in the absence of Far1, though not when Far1 is present. Together, this demonstrates that functionally overlapping regulatory circuits controlling the G1/S transition collectively provide robustness to the G1 arrest response. We also sought to reexamine the phenomenon of pheromone-induced loss of G1/S cyclin proteins, which we suspected could be another Far1-independent arrest mechanism. We confirmed that pheromone treatment has an effect on G1 cyclin protein levels independent of transcriptional control. Our findings suggest that this phenomenon is dependent on SCFGrr1but is at least partly independent of Cdc28 activity, the CDK phosphorylation sites in Cln2, and Far1. We were not, however, able to obtain evidence that pheromone increases the degradation rate of Cln1/2, which raises the possibility that pheromone reduces their synthesis rate instead.
Finally, we also studied the function of Far1 during pheromone-induced G1 arrest. Although it has been assumed that Far1 acts as a G1/S cyclin specific CDK inhibitor, there has been no conclusive evidence that this is the case. Our data, however, suggests that at least part of Far1’s function may actually be to interfere with Cln-CDK/substrate interactions since we saw a significant decrease of co-pulldown of Cln2 and substrates after treatment with pheromone. All together, the results presented here demonstrate that there are numerous independent mechanisms in place to help robustly arrest cells in G1.
Pope, Patricia A., "Investigation of Multiple Concerted Mechanisms Underlying Stimulus-induced G1 Arrest in Yeast: A Dissertation" (2013). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 680.
Available for download on Friday, August 01, 2014