The transcription factor Swi4 is target for PKA regulation of cell size at the G₁ to S transition in Saccharomyces cerevisiae

Abstract

To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G₁, followed by an accumulation of cells in G₂/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G₁ phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G₁ arrest suggesting that Swi4 is a target responsible for PKA modulation of G₁/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4^S159A) and we found that expression of Swi4^S159A protein in the GG104-Swi4Δ strain did not restore the transient G₁ arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G₁/S phase transition.

Keywords:

• budding yeast
• Cln1
• Cyclic AMP
• G₁ cyclins
• Mitosis
• SBF

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Joris Winderickx for providing GG104 strain, Simonetta Piatti for PKBlURA3 plasmid and David Levin for p2713 plasmid bearing the wild type SWI4 gene.

Funding

This work was partially supported by CE project UniCellSys and by the Program SysBioNet, Italian Roadmap research Infrasctructure.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.