A refined two-hybrid system reveals that SCF(Cdc4)-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry.
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.
Kishi
Tsutomu
T
Kishi Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. tkishi@riken.jp
Ikeda
Akemi
A
Koyama
Noriko
N
Fukada
Junji
J
Nagao
Rina
R
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
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
Proc Natl Acad Sci U S A. 2007 Oct 30;104(44):17418-23
17954914
J Biol Chem. 1998 Oct 23;273(43):27757-60
9774381
Genes Dev. 2000 Jul 1;14(13):1595-604
10887154
Genes Dev. 1993 Mar;7(3):517-28
8449406
Nature. 2001 Nov 29;414(6863):514-21
11734846
Nature. 1989 Jul 20;340(6230):245-6
2547163
Mol Cell Biol. 1997 Jun;17(6):3094-102
9154808
Eukaryot Cell. 2003 Jun;2(3):431-45
12796288
Science. 1997 Oct 17;278(5337):455-60
9334303
Cell. 1991 Aug 23;66(4):743-58
1652372
Mol Biol Cell. 2000 Mar;11(3):915-27
10712509
Cell. 1996 Aug 9;86(3):453-63
8756727
Genes Dev. 2001 May 1;15(9):1078-92
11331604
Cell. 1991 Sep 6;66(5):1015-26
1832336
Cell. 2001 Sep 21;106(6):697-708
11572776
Biochim Biophys Acta. 2004 Nov 29;1695(1-3):133-70
15571813
Cell. 1991 Sep 6;66(5):995-1013
1832338
Mol Cell. 2000 Mar;5(3):501-11
10882135
Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6593-8
11381105
Mol Cell Biol. 1999 May;19(5):3312-27
10207056
Nat Biotechnol. 2004 Jul;22(7):888-92
15208639
Mol Cell. 1998 Dec;2(6):709-18
9885559
Mol Biol Cell. 2007 Feb;18(2):426-40
17108329
Cell. 1997 Oct 17;91(2):209-19
9346238
Curr Biol. 2003 Jan 8;13(1):R31-8
12526763
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5187-92
9560251
Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8532-7
9671712
J Cell Sci. 1998 Dec 18;111 ( Pt 24):3655-61
9819356
Mol Biol Cell. 1998 Sep;9(9):2393-405
9725902
Cell. 1994 Oct 21;79(2):233-44
7954792
Annu Rev Biochem. 1998;67:425-79
9759494
Cell. 1987 May 22;49(4):549-58
3552251
Genes Dev. 2003 Jul 15;17(14):1789-802
12865300
Cell. 1997 Oct 17;91(2):221-30
9346239
Oncogene. 2005 Apr 18;24(17):2746-55
15838511