The Start point is a major cell cycle checkpoint in yeast, known as the restriction point in multicellular organisms. The Start checkpoint ensures cell-cycle entry even if conditions later become unfavorable. The physiological factors that control passage through the Start checkpoint include external nutrient concentrations, presence of mating factor/ pheromone, forms of stress, and size control.
Early characterization of Start In an effort to study the ordered events of the cell cycle, Leland Hartwell et al. screened for and characterized temperature sensitive mutants, also known as cell division cycle mutants (cdc mutants), that display arrested cellular development at various stages of the cycle. Hartwell not only identified the mutant, cdc28, which arrests in very early stages of the cell cycle, but he also recognised that the presence of mating factors could result in similar phenotypes of inhibited bud formation and lack of DNA synthesis. Notably, cells that were exposed to mating factors at later stages of the cycle continued division, and only arrested when the resulting daughter cells reached the "early stages" (or more technically, the G1 phase) of the cell cycle. These results suggest that both cdc28 and mating pheromones mediate such early events, and further suggest that there exists a point in the cell cycle where the cell commits to division rather than to mating. Hartwell named this point "Start", where cells are sensitive to mating pheromones prior to reaching this stage, but insensitive to mating factors afterwards. In the years following Hartwell's labor-intensive experiments, it has been shown that other environmental factors contribute to cellular fate in yeast and analogously in other organisms. Though not yeast-specific, a critical study put forth by Zetterberg et al. in 1985 provided evidence for a commitment point in Swiss 3T3 cells, or mouse embryo fibroblasts, when grown in serum-rich or serum-starved conditions. Like the response to mating pheromones in Hartwell's experiments, the response to serum starvation was not uniform amongst all cells. Only postmitotic cells younger than three hours arrested cellular division in these conditions, while cells older than four hours were insensitive to the absence of growth factors. These experimental results show strong evidence for a commitment point to enter mitosis, and consequently suggest that the cell is capable of sensing its environment for cues like growth factors before committing.
Transcription of G1/S genes The transcription of several G1/S genes is essential for cells to proceed through the cell cycle. In budding yeast, the transcription of over 200 genes is activated at the G1/S transition. The transcription of these G1/S genes is primarily regulated by two gene regulatory proteins, SBF and MBF. These regulatory proteins form complexes with SCB and MCB, respectively, which are located on the promoters of G1/S genes.
SBF and MBF regulatory proteins The SBF and MBF complexes are able to activate G1/S transcription only if an inhibitor protein known as Whi5 is dissociated. The dissociation of Whi5 requires phosphorylation by a Cln3-Cdk1 complex. This indicates that the activity of Cln3-Cdk1 plays an important role in the Start checkpoint because of its necessity to simultaneously activate both SBF and MBF proteins. The activity of Cln3 correlates with cell growth rate.
Activation of S-Cdks by G1/S-Cdks G1/S genes include the cyclins Cln1 and Cln2, which can form active complexes with Cdk1. These activated Cln-Cdk complexes help activate S-Cdk complexes, which are normally inhibited by Sic1. Sic1 has no effect on the Cln-Cdk complexes. The Cln-Cdk complexes activate the S-Cdk complexes through the destruction of Sic1 by phosphorylation and subsequent SCF ubiquitination.
Mating factor/ pheromone
Protein interactions between mating pathway and cell cycle progression The response to mating pheromones as described in Hartwell's experiments is unsurprising considering the antagonistic biochemical interactions between the mating pathway and the G1 cyclins that promote cell cycle progression. As shown in the accompanying figure, the mating pathway consists of a MAPK (mitogen-activated protein kinase) cascade, where Ste5 intermediates the pheromone signal and the downstream kinase responses by Ste11, Ste7, and Fus3. From its downstream effects and even immediate ones, Fus3 ultimately activates Far1, which directly inhibits the activity of the G1 cyclins, Cln1/2. In turn, Cln1/2 directly inhibits the mating pathway via Far1 and Ste5 inhibition. The activity of Cln1/2 is mediated by activation of a more upstream G1 cyclin, Cln3. Cln3, along with the cyclin-dependent kinase Cdc28, inactivates and promotes the export of the nuclear Whi5. The export of Whi5 results in the partial activation of the transcription factors SBF and MBF, which ultimately promote cell cycle progression. These transcription factors promote Cln1/2 expression, and enhance the cell cycle response by forming a positive feedback loop, as Cln1/2 promotes SBF activation and Whi5 export.
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