Cell cycle-regulated phosphorylation of Swi6 controls its nuclear localization

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Swi6 is a transcription factor crucial for G1/S-specific gene expression in Saccharomyces cerevisiae.
• Swi6 is known to be highly phosphorylated in vivo during the cell cycle.

Purpose of the Study:

• To investigate the role of Swi6 phosphorylation in regulating its function and localization during the cell cycle.
• To identify the specific phosphorylation sites on Swi6 that change during the cell cycle and their functional significance.

Main Methods:

• Peptide analysis to identify phosphorylation sites on Swi6.
• Site-directed mutagenesis (serine to aspartate or alanine substitutions) to assess the impact of phosphorylation.
• Green fluorescent protein (GFP) fusions to track Swi6 localization.
• Cell cycle synchronization and analysis.

Main Results:

• Serine 160 is identified as the primary cell cycle-regulated phosphorylation site on Swi6.
• Phosphorylation of serine 160 is not dependent on Cdc28 and does not regulate G1/S transcription.
• Phosphorylation of serine 160 controls Swi6's subcellular localization, with phosphorylation promoting cytoplasmic retention and hypophosphorylation enabling nuclear entry.
• Mutations mimicking constitutive phosphorylation (Asp substitution) inhibit nuclear localization, while mutations preventing phosphorylation (Ala substitution) allow nuclear entry throughout the cell cycle.

Conclusions:

• Cell cycle-regulated phosphorylation of Swi6 at serine 160 is a key mechanism controlling its subcellular localization, not its transcriptional activity.
• Swi6 localizes to the nucleus during G1 when hypophosphorylated and to the cytoplasm when phosphorylated from late G1 to late M phase.
• Understanding Swi6 phosphorylation provides insights into cell cycle regulation and transcription factor dynamics.