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Functional overlap among distinct G1/S inhibitory pathways allows robust G1 arrest by yeast mating pheromones.

Pope PA, Pryciak PM - Mol. Biol. Cell (2013)

Bottom Line: In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1.Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways.Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605.

ABSTRACT
In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This route likely involves pheromone-induced degradation of Tec1, a transcriptional activator of the cyclin CLN1, because Tec1 stabilization also causes partial G1 escape in far1 cln2 cells, and this is additive with Whi5/Stb1 removal. Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways. Of interest, although far1 cln2 sic1 cells escaped G1 arrest, they lost viability during pheromone exposure, indicating that G1 exit is deleterious if the arrest signal remains active. Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

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Far1-independent arrest and cell cycle commitment in synchronous cultures. (A) Example of synchronous cell cycle progression and G1 arrest. A PGAL1-CDC20 strain was arrested in M phase (by transfer to glucose medium) and then released (by return to galactose medium) in the presence or absence of α factor. At the times indicated, DNA content of cells was assayed by flow cytometry. In each histogram, the horizontal axis represents fluorescence, and the vertical dimension shows the number of cells. Bottom, the range of fluorescence values used to calculate the proportion of cells with replicated DNA (percentage 2C) in subsequent figures. This example uses a cln2∆ strain (YPAP165). (B) The ability of α factor to halt cell cycle progression was analyzed for four strains, using the PGAL1-CDC20 method described in A. Graphs show mean ± range (n = 2) for wild-type and far1∆ or mean ± SD (n = 4) for cln2∆ and far1∆ cln2∆ strains.(C) Cell cycle commitment occurs earlier in the absence of Far1. After releasing PGAL1-CDC20 cultures from the M-phase block, aliquots were removed at 15-min intervals and treated with pheromone. At 120 min, cells were scored for whether they had arrested in G1 (unbudded cells) or entered the cell cycle (budded). Graphs show mean ± SEM (n = 5); asterisks indicate points where the difference between far1∆ cln2∆ and cln2∆ was deemed statistically significant (p < 0.025; two-tailed unpaired t test).
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Figure 1: Far1-independent arrest and cell cycle commitment in synchronous cultures. (A) Example of synchronous cell cycle progression and G1 arrest. A PGAL1-CDC20 strain was arrested in M phase (by transfer to glucose medium) and then released (by return to galactose medium) in the presence or absence of α factor. At the times indicated, DNA content of cells was assayed by flow cytometry. In each histogram, the horizontal axis represents fluorescence, and the vertical dimension shows the number of cells. Bottom, the range of fluorescence values used to calculate the proportion of cells with replicated DNA (percentage 2C) in subsequent figures. This example uses a cln2∆ strain (YPAP165). (B) The ability of α factor to halt cell cycle progression was analyzed for four strains, using the PGAL1-CDC20 method described in A. Graphs show mean ± range (n = 2) for wild-type and far1∆ or mean ± SD (n = 4) for cln2∆ and far1∆ cln2∆ strains.(C) Cell cycle commitment occurs earlier in the absence of Far1. After releasing PGAL1-CDC20 cultures from the M-phase block, aliquots were removed at 15-min intervals and treated with pheromone. At 120 min, cells were scored for whether they had arrested in G1 (unbudded cells) or entered the cell cycle (budded). Graphs show mean ± SEM (n = 5); asterisks indicate points where the difference between far1∆ cln2∆ and cln2∆ was deemed statistically significant (p < 0.025; two-tailed unpaired t test).

Mentions: In this experimental context (Figure 1A), most cells finish mitosis and enter G1 (i.e., 1C DNA) by 30–60 min after release from the M-phase block and then begin a new round of DNA synthesis roughly 30 min later (at 90 min). If pheromone is added, wild-type cells complete mitosis and then arrest in G1 (for >3 h). To compare multiple different strains and replicate experiments, we plotted the percentage of cells with 2C DNA content as a function of time (Figure 1B). Generally, M-phase–arrested cultures were 80–90% 2C, and by 60 min after release they had cycled back to G1 and were predominantly 1C (i.e., only 10–15% 2C). As expected, far1∆ cells did not arrest in G1 in the presence of pheromone (Figure 1B). (In addition, with or without pheromone, they showed an accelerated return to the 2C state after completing mitosis, consistent with previous findings that Far1 can alter the timing of the G1/S transition even without pheromone treatment; Alberghina et al., 2004.) In contrast, when far1∆ cln2∆ cells were released in the presence of pheromone, they remained in G1 for an extended period (Figure 1B). This arrest was not as strong as in wild-type or FAR1 cln2∆ strains, as evidenced by the gradual increase in cells with 2C DNA content beginning at 120–150 min after release. Thus G1 arrest in the far1∆ cln2∆ cells is partially leaky, but pheromone clearly imposes a durable G1 delay that affects the majority of cells in the culture.


Functional overlap among distinct G1/S inhibitory pathways allows robust G1 arrest by yeast mating pheromones.

Pope PA, Pryciak PM - Mol. Biol. Cell (2013)

Far1-independent arrest and cell cycle commitment in synchronous cultures. (A) Example of synchronous cell cycle progression and G1 arrest. A PGAL1-CDC20 strain was arrested in M phase (by transfer to glucose medium) and then released (by return to galactose medium) in the presence or absence of α factor. At the times indicated, DNA content of cells was assayed by flow cytometry. In each histogram, the horizontal axis represents fluorescence, and the vertical dimension shows the number of cells. Bottom, the range of fluorescence values used to calculate the proportion of cells with replicated DNA (percentage 2C) in subsequent figures. This example uses a cln2∆ strain (YPAP165). (B) The ability of α factor to halt cell cycle progression was analyzed for four strains, using the PGAL1-CDC20 method described in A. Graphs show mean ± range (n = 2) for wild-type and far1∆ or mean ± SD (n = 4) for cln2∆ and far1∆ cln2∆ strains.(C) Cell cycle commitment occurs earlier in the absence of Far1. After releasing PGAL1-CDC20 cultures from the M-phase block, aliquots were removed at 15-min intervals and treated with pheromone. At 120 min, cells were scored for whether they had arrested in G1 (unbudded cells) or entered the cell cycle (budded). Graphs show mean ± SEM (n = 5); asterisks indicate points where the difference between far1∆ cln2∆ and cln2∆ was deemed statistically significant (p < 0.025; two-tailed unpaired t test).
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Related In: Results  -  Collection

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Figure 1: Far1-independent arrest and cell cycle commitment in synchronous cultures. (A) Example of synchronous cell cycle progression and G1 arrest. A PGAL1-CDC20 strain was arrested in M phase (by transfer to glucose medium) and then released (by return to galactose medium) in the presence or absence of α factor. At the times indicated, DNA content of cells was assayed by flow cytometry. In each histogram, the horizontal axis represents fluorescence, and the vertical dimension shows the number of cells. Bottom, the range of fluorescence values used to calculate the proportion of cells with replicated DNA (percentage 2C) in subsequent figures. This example uses a cln2∆ strain (YPAP165). (B) The ability of α factor to halt cell cycle progression was analyzed for four strains, using the PGAL1-CDC20 method described in A. Graphs show mean ± range (n = 2) for wild-type and far1∆ or mean ± SD (n = 4) for cln2∆ and far1∆ cln2∆ strains.(C) Cell cycle commitment occurs earlier in the absence of Far1. After releasing PGAL1-CDC20 cultures from the M-phase block, aliquots were removed at 15-min intervals and treated with pheromone. At 120 min, cells were scored for whether they had arrested in G1 (unbudded cells) or entered the cell cycle (budded). Graphs show mean ± SEM (n = 5); asterisks indicate points where the difference between far1∆ cln2∆ and cln2∆ was deemed statistically significant (p < 0.025; two-tailed unpaired t test).
Mentions: In this experimental context (Figure 1A), most cells finish mitosis and enter G1 (i.e., 1C DNA) by 30–60 min after release from the M-phase block and then begin a new round of DNA synthesis roughly 30 min later (at 90 min). If pheromone is added, wild-type cells complete mitosis and then arrest in G1 (for >3 h). To compare multiple different strains and replicate experiments, we plotted the percentage of cells with 2C DNA content as a function of time (Figure 1B). Generally, M-phase–arrested cultures were 80–90% 2C, and by 60 min after release they had cycled back to G1 and were predominantly 1C (i.e., only 10–15% 2C). As expected, far1∆ cells did not arrest in G1 in the presence of pheromone (Figure 1B). (In addition, with or without pheromone, they showed an accelerated return to the 2C state after completing mitosis, consistent with previous findings that Far1 can alter the timing of the G1/S transition even without pheromone treatment; Alberghina et al., 2004.) In contrast, when far1∆ cln2∆ cells were released in the presence of pheromone, they remained in G1 for an extended period (Figure 1B). This arrest was not as strong as in wild-type or FAR1 cln2∆ strains, as evidenced by the gradual increase in cells with 2C DNA content beginning at 120–150 min after release. Thus G1 arrest in the far1∆ cln2∆ cells is partially leaky, but pheromone clearly imposes a durable G1 delay that affects the majority of cells in the culture.

Bottom Line: In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1.Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways.Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605.

ABSTRACT
In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This route likely involves pheromone-induced degradation of Tec1, a transcriptional activator of the cyclin CLN1, because Tec1 stabilization also causes partial G1 escape in far1 cln2 cells, and this is additive with Whi5/Stb1 removal. Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways. Of interest, although far1 cln2 sic1 cells escaped G1 arrest, they lost viability during pheromone exposure, indicating that G1 exit is deleterious if the arrest signal remains active. Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

Show MeSH
Related in: MedlinePlus