Reconstitution of G1 Cyclin Ubiquitination with Complexes Containing SCFGrr1 and Rbx1
Dorota Skowyra,
1
Deanna M. Koepp,
123
Takumi Kamura,
45
Michael N. Conrad,
5
Ronald C. Conaway,
5
Joan Weliky Conaway,
456
Stephen
J. Elledge,
123
J.
Wade Harper
1*
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.
1 Verna and Marrs McLean Department of
Biochemistry,
2 Department of Molecular and Human
Genetics,
3 Howard Hughes Medical Institute, Baylor
College of Medicine, Houston, TX 77030, USA.
4 Howard Hughes Medical Institute and
5 Program in Molecular and Cell Biology, Oklahoma
Medical Research Foundation, Oklahoma City, OK 73104, USA.
6 Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
*
To whom correspondence should be addressed. E-mail:
jharper{at}bcm.tmc.edu.
