Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1.
Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.
Skowyra
D
D
Verna and Marrs McLean Department of Biochemistry, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.
Koepp
D M
DM
Kamura
T
T
Conrad
M N
MN
Conaway
R C
RC
Conaway
J W
JW
Elledge
S J
SJ
Harper
J W
JW
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D044842:Cullin Proteins; D016213:Cyclins / Q000378:metabolism*; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
C082719:CLN1 protein, S cerevisiae; D002352:Carrier Proteins; C100988:Cdc53 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D016213:Cyclins; D044783:F-Box Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C071032:GRR1 protein, S cerevisiae; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D010453:Peptide Synthases