101. A refined two-hybrid system reveals that SCF(Cdc4)-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry.
- Author
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Kishi T, Ikeda A, Koyama N, Fukada J, and Nagao R
- Subjects
- Cullin Proteins metabolism, Cyclin-Dependent Kinase Inhibitor Proteins, Cyclins genetics, Cyclins metabolism, F-Box Proteins metabolism, G1 Phase, Gene Expression Regulation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Two-Hybrid System Techniques, Ubiquitination, Cell Cycle Proteins metabolism, Fungal Proteins metabolism, S Phase, SKP Cullin F-Box Protein Ligases metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription Factors metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Ubiquitin-dependent degradation is implicated in various cellular regulatory mechanisms. The SCF(Cdc4) (Skp1, Cullin/Cdc53, and the F-box protein Cdc4) complex is an ubiquitin ligase complex that acts as a regulator of cell cycle, signal transduction, and transcription. These regulatory mechanisms are not well defined because of the difficulty in identifying the interaction between ubiquitin ligases and their substrates. To identify substrates of the yeast SCF(Cdc4) ubiquitin ligase complex, we refined the yeast two-hybrid system to allow screening Cdc4-substrate interactions under conditions of substrate stabilization, and identified Swi5 as a substrate of the SCF(Cdc4) complex. Swi5 is the transcriptional activator of Sic1, the inhibitor of S phase cyclin-dependent kinases (CDKs). We showed that Swi5 is indeed ubiquitinated and degraded through the SCF(Cdc4) complex. Furthermore, the SCF(Cdc4)-dependent degradation of Swi5 was required to terminate SIC1 transcription at early G(1) phase, which ensured efficient entry into S phase: Hyperaccumulation of Sic1 was noted in cells expressing stabilized Swi5, and expression of stabilized Swi5 delayed S phase entry, which was dominantly suppressed by SIC1 deletion. These findings indicate that the SCF(Cdc4) complex regulates S phase entry not only through degradation of Sic1, but also through degradation of Swi5.
- Published
- 2008
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