Start point (yeast)

Last updated November 07, 2023

The Start point is a major cell cycle checkpoint in yeast, known as the restriction point in multicellular organisms.^[1] 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.^[2]

Early characterization of Start
Transcription of G1/S genes
SBF and MBF regulatory proteins
Activation of S-Cdks by G1/S-Cdks
Mating factor/ pheromone
Protein interactions between mating pathway and cell cycle progression
Quantitative description of start
Role of Whi5 in cell cycle progression
Molecular dynamics between mating pathway and cell cycle progression
Cell nutrient growth and size control
References
See also

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.^[3] 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.^[4] 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.^[5] 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.^[2]

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.^[2] 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.^[2]

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.^[2] 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 ^[3] 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,^[6] 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.

Quantitative description of start

A modern-day study delineating the relationship between mating arrest and cell cycle progression was put forth by Doncic et al. in June 2011.^[6] Recognizing that the amount of nuclear Whi5 is an indicator of G1 cyclin activity, the authors set out to quantitatively understand the point at which cells commit to division. With a microfluidic platform, an asynchronous population of cells was exposed to the pheromone, alpha-factor. Using a Whi5-GFP fusion protein, they tracked the amount of nuclear Whi5 following the addition of alpha-factor, and noted whether the cell arrested or continued division. As expected pre-Start cells arrested cellular division upon pheromone addition, as indicated by the small fraction of Whi5 export. Conversely, post-Start cells were insensitive to alpha-factor and continued division, as reflected by the large fraction of Whi5 export. Thus, the differential response to the presence of pheromones is reflected in whether the cell is pre- or post-Start, states that can be characterized by how much Whi5 is present in the nucleus. Logistic regression was next used to calculate the probability of arrest relative to the fraction of exported Whi5 and showed a sharp switch between arrest and progression when approximately 50% of the Whi5 was exported from the nucleus. It was also shown that this fraction of exported Whi5 corresponds to the activation of the Cln1/2 positive feedback loop (see below). In conclusion this indicates that that Start is defined by the activation of the Cln1/2 feedback loop.

Role of Whi5 in cell cycle progression

As mentioned above, the G1 cyclins, Cln1/2, are part of a positive feedback loop that promotes their own transcription and the activation of SBF and MBF transcription factors. In 2008, Skotheim et al. proposed that this feedback loop allows for a strong signal to commit to cellular division by the SBF and MBF regulated genes.^[5] They hypothesized that without a coherent expression of the genes necessary for early events, like DNA replication and bud-site formation, random individual cellular signals creates noise that weakens the commitment response. Noting the long and asynchronous induction times of CLN2 and RAD27 (a gene in the SBF/MBF regulon) in cln1∆cln2∆ cells as compared to wild type, Skotheim et al. thus concluded that the Cln1/2 positive feedback mechanism allows for a synchronous and more efficient expression of the SBF/MBF regulon.

The authors further observed that Whi5 phosphorylation and consequent inactivation plays a role in this positive feedback response. A Whi5 allele lacking six of twelve phosphorylation sites results in a slow exit from the nucleus, and consequently a less coherent induction of CLN2 and RAD27 expression. Thus, the inability to phosphorylate Whi5 disrupts the Cln1/2 positive feedback loop, and in turn reduces the coherent regulon expression.

Molecular dynamics between mating pathway and cell cycle progression

To further elucidate a biochemical explanation between mating arrest and cell cycle commitment, Doncic et al. conducted the same commitment assay described above on various mutant strains.^[6] The mutant, FAR1-S87A, lacks CDK phosphorylation sites, and thus Cln1/2 inhibition of Far1 is compromised. The result is an increase in the amount of Whi5 export required to commit to cellular division, suggesting that Far 1 phosphorylation is key to cellular commitment. Conversely, the mutant, STE5-8A, lacking CDK phosphorylation sites as well (and thus Cln1/2 inhibition of Ste5 is compromised), does not shift the commitment point, suggesting that such inhibition of the mating pathway is not critical for Start. A further time-lapse analysis of STE5-8A cells reveals that these mutant cells cannot fully commit to cellular division, as cells exposed to alpha-factor will bud and then revert to mating without completing the cell cycle. Doncic et al. proposed that the incomplete division was due to expression of genes in both the mating pathway and in the G1 cyclin-driven cellular progression. Indeed, tracking the expression of FUS1pr-GFP, a mating pathway gene, and of CLN2pr-mCherry, a cell cycle gene, showed great coexpression in STE5-8A cells relative to wild type cells.

Thus, Cln1/2 inhibition of Far1 allows for entry into the cell cycle (Start), while inhibition of Ste5 guarantees distinct expression of genes for either the mating pathway or for cell cycle progression.

Cell nutrient growth and size control

The external nutrient concentrations are extremely important to proceeding through the Start checkpoint. The availability of nutrients is strongly correlated to cell growth size. Cells will not proceed if they do not reach a certain size due to nutrient deprivation, usually nitrogen. Thus, larger cells spend less time in the Start checkpoint compared to smaller cells.^[2]

Related Research Articles

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that causes it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm, chromosomes and other components into two daughter cells in a process called cell division.

Anaphase-promoting complex is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin (Apc2) and RING (Apc11) subunit much like SCF. Other parts of the APC/C have unknown functions but are highly conserved.

A cyclin-dependent kinase complex is a protein complex formed by the association of an inactive catalytic subunit of a protein kinase, cyclin-dependent kinase (CDK), with a regulatory subunit, cyclin. Once cyclin-dependent kinases bind to cyclin, the formed complex is in an activated state. Substrate specificity of the activated complex is mainly established by the associated cyclin within the complex. Activity of CDKCs is controlled by phosphorylation of target proteins, as well as binding of inhibitory proteins.

S phase (Synthesis phase) is the phase of the cell cycle in which DNA is replicated, occurring between G₁ phase and G₂ phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.

The restriction point (R), also known as the Start or G₁/S checkpoint, is a cell cycle checkpoint in the G₁ phase of the animal cell cycle at which the cell becomes "committed" to the cell cycle, and after which extracellular signals are no longer required to stimulate proliferation. The defining biochemical feature of the restriction point is the activation of G₁/S- and S-phase cyclin-CDK complexes, which in turn phosphorylate proteins that initiate DNA replication, centrosome duplication, and other early cell cycle events. It is one of three main cell cycle checkpoints, the other two being the G2-M DNA damage checkpoint and the spindle checkpoint.

Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.

The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is governed by cell cycle checkpoints to ensure cell cycle integrity and the subsequent S phase can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated checkpoint can lead to cellular transformation and disease states such as cancer.

Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages.

CDK-activating kinase (CAK) activates the cyclin-CDK complex by phosphorylating threonine residue 160 in the CDK activation loop. CAK itself is a member of the Cdk family and functions as a positive regulator of Cdk1, Cdk2, Cdk4, and Cdk6.

<span class="mw-page-title-main">Cyclin D</span> Member of the cyclin protein family

Cyclin D is a member of the cyclin protein family that is involved in regulating cell cycle progression. The synthesis of cyclin D is initiated during G1 and drives the G1/S phase transition. Cyclin D protein is anywhere from 155 to 477 amino acids in length.

Cyclin-dependent kinase 2, also known as cell division protein kinase 2, or Cdk2, is an enzyme that in humans is encoded by the CDK2 gene. The protein encoded by this gene is a member of the cyclin-dependent kinase family of Ser/Thr protein kinases. This protein kinase is highly similar to the gene products of S. cerevisiae cdc28, and S. pombe cdc2, also known as Cdk1 in humans. It is a catalytic subunit of the cyclin-dependent kinase complex, whose activity is restricted to the G1-S phase of the cell cycle, where cells make proteins necessary for mitosis and replicate their DNA. This protein associates with and is regulated by the regulatory subunits of the complex including cyclin E or A. Cyclin E binds G1 phase Cdk2, which is required for the transition from G1 to S phase while binding with Cyclin A is required to progress through the S phase. Its activity is also regulated by phosphorylation. Multiple alternatively spliced variants and multiple transcription initiation sites of this gene have been reported. The role of this protein in G1-S transition has been recently questioned as cells lacking Cdk2 are reported to have no problem during this transition.

Cyclin-dependent kinase 1 also known as CDK1 or cell division cycle protein 2 homolog is a highly conserved protein that functions as a serine/threonine protein kinase, and is a key player in cell cycle regulation. It has been highly studied in the budding yeast S. cerevisiae, and the fission yeast S. pombe, where it is encoded by genes cdc28 and cdc2, respectively. With its cyclin partners, Cdk1 forms complexes that phosphorylate a variety of target substrates ; phosphorylation of these proteins leads to cell cycle progression.

G1/S-specific cyclin Cln3 is a protein that is encoded by the CLN3 gene. The Cln3 protein is a budding yeast G1 cyclin that controls the timing of Start, the point of commitment to a mitotic cell cycle. It is an upstream regulator of the other G1 cyclins, and it is thought to be the key regulator linking cell growth to cell cycle progression. It is a 65 kD, unstable protein; like other cyclins, it functions by binding and activating cyclin-dependent kinase (CDK).

Sic1, a protein, is a stoichiometric inhibitor of Cdk1-Clb complexes in the budding yeast Saccharomyces cerevisiae. Because B-type cyclin-Cdk1 complexes are the drivers of S-phase initiation, Sic1 prevents premature S-phase entry. Multisite phosphorylation of Sic1 is thought to time Sic1 ubiquitination and destruction, and by extension, the timing of S-phase entry.

A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.

Cln1, Cln2, and Cln3 are cyclin proteins expressed in the G1-phase of the cell cycle of budding yeast. Like other cyclins, they function by binding and activating cyclin-dependent kinase. They are responsible for initiating entry into a new mitotic cell cycle at Start. As described below, Cln3 is the primary regulator of this process during normal yeast growth, with the other two G1 cyclins performing their function upon induction by Cln3. However, Cln1 and Cln2 are also directly regulated by pathways sensing extracellular conditions, including the mating pheremone pathway.

Clb5 and Clb6 are B-type, S-phase cyclins in yeast that assist in cell cycle regulation. Clb5 and Clb6 bind and activate Cdk1, and high levels of these cyclins are required for entering S-phase. S-phase cyclin binding to Cdk1 directly stimulates DNA replication as well as progression to the next phase of the cell cycle.

Fus3 is a MAPK protein involved in the mating decision of yeast. The dissociation of Fus3 from scaffold protein Ste5 results in the switch-like mating decision observed in yeast. During this process, Fus3 competes with a phosphatase Ptc1, attempting to phosphorylate 4 key phosphorylation sites on Ste5. When all 4 sites on Ste5 have been dephosphorylated by Ptc1, Fus3 dissociates from Ste5 and trans locates to the nucleus.

Whi5 is a transcriptional regulator in the budding yeast cell cycle, notably in the G1 phase. It is an inhibitor of SBF, which is involved in the transcription of G1-specific genes. Cln3 promotes the disassociation of Whi5 from SBF, and its disassociation results in the transcription of genes needed to enter S phase.

BCK2, also named CTR7, is an early cell cycle regulator expressed by the yeast Saccharomyces cerevisiae. It was first discovered in a screen for genes whose overexpression would suppress the phenotypes of PKC1 pathway mutations. Though its mechanism is currently unknown, it is believed to interact with Swi4 and Mcm1, both important transcriptional regulators of early cell cycle.

References

↑ Irvali, D; Schlottmann, FP; Muralidhara, P; Nadelson, I; Kleemann, K; Wood, NE; Doncic, A; Ewald, JC (16 January 2023). "When yeast cells change their mind: cell cycle "Start" is reversible under starvation". The EMBO Journal. 42 (2): e110321. doi:10.15252/embj.2021110321. PMC 9841329 . PMID 36420556.
1 2 3 4 5 6 Morgan, David. The Cell Cycle: Principles of Cell Control. New Science Press Ltd., London, 2007; pp 196-203.
1 2 Hartwell, L. H. "Genetic Control of the Cell-Division Cycle in Yeast, I. Detection of Mutants." Proceedings of the National Academy of Sciences 66.2 (1970): 352-59.
↑ Zetterberg, A., and Olle Larsson. "Kinetic Analysis of Regulatory Events in G1 Leading to Proliferation or Quiescence of Swiss 3T3 Cells." PNAS 82 (1985): 5365-369.
1 2 Skotheim, J.; DiTalia, S.; ED Siggia , E.; Cross, F. Positive feedback of G1 cyclins ensures coherent cell cycle entry. Nature 454, 291-296 (2008).
1 2 3 Doncic, Andreas, Melody Falleur-Fettig, and Jan M. Skotheim. "Distinct Interactions Select and Maintain a Specific Cell Fate." Molecular Cell 43.4 (2011): 528-39.