A Refined Two-Hybrid System Reveals That${\rm SCF}^{{\rm Cdc}4}\text{-Dependent}$Degradation of Swi5 Contributes to the Regulatory Mechanism of S-Phase Entry

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 38; pp. 14497 - 14502
• Main Authors: Kishi, Tsutomu, Ikeda, Akemi, Koyama, Noriko, Fukada, Junji, Nagao, Rina
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences 23.09.2008
• Subjects: Cell cycle; Cell cycle proteins; Genes; Interphase; Metaphase; Phosphorylation; Plasmids; Transcriptional regulatory elements; Ubiquitins; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0806253105

Ubiquitin-dependent degradation is implicated in various cellular regulatory mechanisms. The ${\rm SCF}^{{\rm Cdc}4}$ (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 ${\rm SCF}^{{\rm Cdc}4}$ 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 ${\rm SCF}^{{\rm Cdc}4}$ 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 ${\rm SCF}^{{\rm Cdc}4}$ complex. Furthermore, the ${\rm SCF}^{{\rm Cdc}4}\text{-dependent}$ degradation of Swi5 was required to terminate SIC1 transcription at early G₁ 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 ${\rm SCF}^{{\rm Cdc}4}$ complex regulates S phase entry not only through degradation of Sic1, but also through degradation of Swi5.