Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.
SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.
Seol
J H
JH
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Feldman
R M
RM
Zachariae
W
W
Shevchenko
A
A
Correll
C C
CC
Lyapina
S
S
Chi
Y
Y
Galova
M
M
Claypool
J
J
Sandmeyer
S
S
Nasmyth
K
K
Deshaies
R J
RJ
Shevchenko
A
A
Deshaies
R J
RJ
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D064198:Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044842:Cullin Proteins; D004789:Enzyme Activation; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D064173:Anaphase-Promoting Complex-Cyclosome; D064198:Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome; D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; D008025:Ligases; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins; C100988:Cdc53 protein, S cerevisiae; C513273:FBXW7 protein, human; C485631:HEY1 protein, human; C577449:CDC34 protein, S cerevisiae; C577447:CDC34 protein, human; C577351:ANAPC11 protein, human; C485632:HRT1 protein, S cerevisiae