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Functional studies of the yeast med5, med15 and med16 mediator tail subunits.

Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S - PLoS ONE (2013)

Bottom Line: We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle.Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype.Collectively, these findings provide insight into the function of the Mediator Tail module.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

ABSTRACT
The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature-sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when Med5/15 or Med15/16 are inactivated together. We identify 25 genes in each double mutant that show a significant change in expression when compared to the corresponding single mutants and to the wild type strain. Importantly, 13 of the 25 identified genes are common for both double mutants. We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle. Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype. Collectively, these findings provide insight into the function of the Mediator Tail module.

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Double deletions of yeast MED5/MED15 and MED15/MED16 are synthetically lethal.(A) The strains indicated to the left were plated as 10-fold serial dilutions, 5 µl spots at permissive conditions (24°C, Cu++, glucose) and restrictive conditions (37°C, no Cu++, galactose) respectively. (B) The strains indicated to the right were grown in liquid media, at restrictive conditions, for 24 hours. OD600 was measured at the indicated time points. OD600 at 0 hours was set to 1 and the other values where normalized against time = 0 min. The graphs represent the mean of two separate experiments. The graphs represent the mean of two separate experiments, and the error bars represent the standard deviation.
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pone-0073137-g002: Double deletions of yeast MED5/MED15 and MED15/MED16 are synthetically lethal.(A) The strains indicated to the left were plated as 10-fold serial dilutions, 5 µl spots at permissive conditions (24°C, Cu++, glucose) and restrictive conditions (37°C, no Cu++, galactose) respectively. (B) The strains indicated to the right were grown in liquid media, at restrictive conditions, for 24 hours. OD600 was measured at the indicated time points. OD600 at 0 hours was set to 1 and the other values where normalized against time = 0 min. The graphs represent the mean of two separate experiments. The graphs represent the mean of two separate experiments, and the error bars represent the standard deviation.

Mentions: We and others have previously reported that single disruptions of MED5, MED15 or MED16 are viable, in contrast to double deletions of MED5 and MED15 or MED15 and MED16, which all cause synthetic lethality [26], [27]. However, because the cells carrying the double deletions do not grow it has not previously been possible to study the cause of lethality. In order to test if we could reproduce the synthetic lethality in a controlled, heat-inducible way using the N-Degron system, we grew wild type cells, strains expressing individual Med5, Med15 or Med16 N-Degron constructs and strains that express N-Degron-tagged versions of both Med5/Med15 and Med15/Med16, respectively. The cells were initially grown in YPD at 24°C (permissive conditions), then transferred either to YPG-plates (Figure 2A) or liquid YPG-media (Figure 2B) and moved to 37°C (restrictive conditions). When the cells were grown on plates, degradation of Med5 alone had little or no effect on growth, while single degradation of Med16 showed a mild growth defect, and single degradation of Med15 showed a more pronounced effect on growth. This limited growth effect for a med15 mutant has been reported previously [27]. In contrast, we found that both the med5/med15 and the med15/med16 double Degron strains were unable to grow on plates at the restrictive conditions (Figure 2A). These results indicate that the N-Degron system can be used to reproduce the effects of ts mutants in the tail module that have been created using conventional methods. Similar results were obtained in liquid media (Figure 2B).


Functional studies of the yeast med5, med15 and med16 mediator tail subunits.

Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S - PLoS ONE (2013)

Double deletions of yeast MED5/MED15 and MED15/MED16 are synthetically lethal.(A) The strains indicated to the left were plated as 10-fold serial dilutions, 5 µl spots at permissive conditions (24°C, Cu++, glucose) and restrictive conditions (37°C, no Cu++, galactose) respectively. (B) The strains indicated to the right were grown in liquid media, at restrictive conditions, for 24 hours. OD600 was measured at the indicated time points. OD600 at 0 hours was set to 1 and the other values where normalized against time = 0 min. The graphs represent the mean of two separate experiments. The graphs represent the mean of two separate experiments, and the error bars represent the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750046&req=5

pone-0073137-g002: Double deletions of yeast MED5/MED15 and MED15/MED16 are synthetically lethal.(A) The strains indicated to the left were plated as 10-fold serial dilutions, 5 µl spots at permissive conditions (24°C, Cu++, glucose) and restrictive conditions (37°C, no Cu++, galactose) respectively. (B) The strains indicated to the right were grown in liquid media, at restrictive conditions, for 24 hours. OD600 was measured at the indicated time points. OD600 at 0 hours was set to 1 and the other values where normalized against time = 0 min. The graphs represent the mean of two separate experiments. The graphs represent the mean of two separate experiments, and the error bars represent the standard deviation.
Mentions: We and others have previously reported that single disruptions of MED5, MED15 or MED16 are viable, in contrast to double deletions of MED5 and MED15 or MED15 and MED16, which all cause synthetic lethality [26], [27]. However, because the cells carrying the double deletions do not grow it has not previously been possible to study the cause of lethality. In order to test if we could reproduce the synthetic lethality in a controlled, heat-inducible way using the N-Degron system, we grew wild type cells, strains expressing individual Med5, Med15 or Med16 N-Degron constructs and strains that express N-Degron-tagged versions of both Med5/Med15 and Med15/Med16, respectively. The cells were initially grown in YPD at 24°C (permissive conditions), then transferred either to YPG-plates (Figure 2A) or liquid YPG-media (Figure 2B) and moved to 37°C (restrictive conditions). When the cells were grown on plates, degradation of Med5 alone had little or no effect on growth, while single degradation of Med16 showed a mild growth defect, and single degradation of Med15 showed a more pronounced effect on growth. This limited growth effect for a med15 mutant has been reported previously [27]. In contrast, we found that both the med5/med15 and the med15/med16 double Degron strains were unable to grow on plates at the restrictive conditions (Figure 2A). These results indicate that the N-Degron system can be used to reproduce the effects of ts mutants in the tail module that have been created using conventional methods. Similar results were obtained in liquid media (Figure 2B).

Bottom Line: We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle.Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype.Collectively, these findings provide insight into the function of the Mediator Tail module.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

ABSTRACT
The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature-sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when Med5/15 or Med15/16 are inactivated together. We identify 25 genes in each double mutant that show a significant change in expression when compared to the corresponding single mutants and to the wild type strain. Importantly, 13 of the 25 identified genes are common for both double mutants. We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle. Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype. Collectively, these findings provide insight into the function of the Mediator Tail module.

Show MeSH
Related in: MedlinePlus