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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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Confirmation of expression and specific, induced degradation of the MED5, MED15 and MED16 Degron constructs.(A) Crude protein extracts isolated from the wild type strain and each of the med5, med15, and med16 Degron strains, grown at the permissive conditions (24°C/YPD/0.1 mM CuSO4), were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with α-Med5, α-Med15 and α-Med16 antibodies, respectively. (B) Crude protein extracts were isolated from the wild type strain and each of the med5, med15 and med16 Degron strains at the permissive conditions (uninduced, u.i.) and 45 minutes after switching to the non-permissive growth conditions. The extracts were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with anti c-myc antibodies (specific for the Degron-tag). (C) Specific degradation of Degron-tagged MED5. Crude protein extracts were isolated from the wild type strain, the med5 Degron strain, and the med15 Degron strain 45 minutes after switching to the restrictive growth conditions. Proteins were separated on 10% SDS-PAGE, transferred to PVDF-membranes and blotted with α-Med5 antibodies.
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pone-0073137-g001: Confirmation of expression and specific, induced degradation of the MED5, MED15 and MED16 Degron constructs.(A) Crude protein extracts isolated from the wild type strain and each of the med5, med15, and med16 Degron strains, grown at the permissive conditions (24°C/YPD/0.1 mM CuSO4), were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with α-Med5, α-Med15 and α-Med16 antibodies, respectively. (B) Crude protein extracts were isolated from the wild type strain and each of the med5, med15 and med16 Degron strains at the permissive conditions (uninduced, u.i.) and 45 minutes after switching to the non-permissive growth conditions. The extracts were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with anti c-myc antibodies (specific for the Degron-tag). (C) Specific degradation of Degron-tagged MED5. Crude protein extracts were isolated from the wild type strain, the med5 Degron strain, and the med15 Degron strain 45 minutes after switching to the restrictive growth conditions. Proteins were separated on 10% SDS-PAGE, transferred to PVDF-membranes and blotted with α-Med5 antibodies.

Mentions: We adopted the N-Degron system to obtain copper-dependent expression as well as galactose- and temperature-dependent degradation of the Med5, Med15 and Med16 Mediator subunits, either individually or pairwise [28]. All Degron constructs were expressed from their normal chromosomal location under the control of their respective endogenous promoters. In order to study the expression of the Degron-tagged proteins, we prepared whole-cell protein extracts from wild type cells and from each single Degron strain. The extracts were analyzed by Western blotting using antibodies specific for each protein (Figure 1A). We found that the Degron-tagged Med5, Med15 and Med16 were all expressed at levels similar to the levels of each protein in the parental wild type strain. When comparing the levels of each Degron-tagged protein, before and after heat-induction, we found that each Degron-tagged protein was efficiently depleted from the extracts already at 45 minutes after heat-induction (Figure 1B). Finally, we also tested that the heat-degradation of Med5 was specific to the strain expressing the Degron-tagged version. We found that untagged Med5 is stable in both the wild type strain and in the strain that expresses a Degron-tagged version of Med15, but degraded in the strain that expresses the Degron-tagged Med5 (Figure 1C). Thus, we conclude that the mutant strains encoding Degron-tagged versions of Med5, Med15 and Med16 function properly.


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)

Confirmation of expression and specific, induced degradation of the MED5, MED15 and MED16 Degron constructs.(A) Crude protein extracts isolated from the wild type strain and each of the med5, med15, and med16 Degron strains, grown at the permissive conditions (24°C/YPD/0.1 mM CuSO4), were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with α-Med5, α-Med15 and α-Med16 antibodies, respectively. (B) Crude protein extracts were isolated from the wild type strain and each of the med5, med15 and med16 Degron strains at the permissive conditions (uninduced, u.i.) and 45 minutes after switching to the non-permissive growth conditions. The extracts were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with anti c-myc antibodies (specific for the Degron-tag). (C) Specific degradation of Degron-tagged MED5. Crude protein extracts were isolated from the wild type strain, the med5 Degron strain, and the med15 Degron strain 45 minutes after switching to the restrictive growth conditions. Proteins were separated on 10% SDS-PAGE, transferred to PVDF-membranes and blotted with α-Med5 antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750046&req=5

pone-0073137-g001: Confirmation of expression and specific, induced degradation of the MED5, MED15 and MED16 Degron constructs.(A) Crude protein extracts isolated from the wild type strain and each of the med5, med15, and med16 Degron strains, grown at the permissive conditions (24°C/YPD/0.1 mM CuSO4), were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with α-Med5, α-Med15 and α-Med16 antibodies, respectively. (B) Crude protein extracts were isolated from the wild type strain and each of the med5, med15 and med16 Degron strains at the permissive conditions (uninduced, u.i.) and 45 minutes after switching to the non-permissive growth conditions. The extracts were separated on 10% SDS-PAGE, transferred to PVDF membranes and blotted with anti c-myc antibodies (specific for the Degron-tag). (C) Specific degradation of Degron-tagged MED5. Crude protein extracts were isolated from the wild type strain, the med5 Degron strain, and the med15 Degron strain 45 minutes after switching to the restrictive growth conditions. Proteins were separated on 10% SDS-PAGE, transferred to PVDF-membranes and blotted with α-Med5 antibodies.
Mentions: We adopted the N-Degron system to obtain copper-dependent expression as well as galactose- and temperature-dependent degradation of the Med5, Med15 and Med16 Mediator subunits, either individually or pairwise [28]. All Degron constructs were expressed from their normal chromosomal location under the control of their respective endogenous promoters. In order to study the expression of the Degron-tagged proteins, we prepared whole-cell protein extracts from wild type cells and from each single Degron strain. The extracts were analyzed by Western blotting using antibodies specific for each protein (Figure 1A). We found that the Degron-tagged Med5, Med15 and Med16 were all expressed at levels similar to the levels of each protein in the parental wild type strain. When comparing the levels of each Degron-tagged protein, before and after heat-induction, we found that each Degron-tagged protein was efficiently depleted from the extracts already at 45 minutes after heat-induction (Figure 1B). Finally, we also tested that the heat-degradation of Med5 was specific to the strain expressing the Degron-tagged version. We found that untagged Med5 is stable in both the wild type strain and in the strain that expresses a Degron-tagged version of Med15, but degraded in the strain that expresses the Degron-tagged Med5 (Figure 1C). Thus, we conclude that the mutant strains encoding Degron-tagged versions of Med5, Med15 and Med16 function properly.

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