title: A refined two-hybrid system reveals that SCF(Cdc4)-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry.
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.
authors.affiliation: Kishi Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. tkishi@riken.jp
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kishi
authors.forename: Akemi
authors.initials: A
authors.lastname: Ikeda
authors.forename: Noriko
authors.initials: N
authors.lastname: Koyama
authors.forename: Junji
authors.initials: J
authors.lastname: Fukada
authors.forename: Rina
authors.initials: R
authors.lastname: Nagao
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D044842:Cullin Proteins / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D044783:F-Box Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D016193:G1 Phase; D005786:Gene Expression Regulation; D016196:S Phase; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C080443:CDC4 protein, S cerevisiae; C100988:Cdc53 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D044783:F-Box Proteins; D005656:Fungal Proteins; C086926:SIC1 protein, S cerevisiae; C080218:SWI5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D044843:SKP Cullin F-Box Protein Ligases; C120507:SKP1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases
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