Reconstitution of G(1) cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1

Control of cyclin Levels is critical for proper cell cycle regulation. In y east, the stability of the Ci,,cyclin Cln1 is controlled by phosphorylation -dependent ubiquitination. Here it is shown that this reaction can be recon stituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated C ln1 was ubiquitinated by SCF (Skp1Cdc53-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 Hipp e-Lindau tumor suppressor complex, may define a previously unrecognized cla ss of E3-associated proteins.