An essential function of Grr1 for the degradation of Cln2 is to act as a binding core that links Cln2 to Skp1

In budding yeast, SCF complexes, composed of Skp1, Cdc53 and one of the F-b ox proteins, have been implicated in Cdc34-dependent ubiquitination, Grr1, which is required for degradation of G(1) cyclins Cln1 and Cln2 as well as for regulation of glucose repression, is an F-box protein and interacts wit h Skp1 through the F-box motif, Grr1 also interacts in vitro with phosphory lated Cln1. and Cln2, However, ubiquitination of Cln1 has not been successf ul in an in vitro reconstituted system. In this study, domain analysis was performed to understand the role of Grr1 in the degradation of Cln2, Grr1 h as another motif, leucine-rich repeats (LRR), in addition to the F-box, We found that the LRR is a domain for Cln2 binding. A deletion of half of the LRR abolished the interaction of Grr1 with phosphorylated Cln2 but not with Skp1 in vivo, and a deletion of the F-box abolished the interaction of Grr 1 with Skp1 but not with phosphorylated Cln2 in vivo, Based on these result s, we constructed grr1 mutants that are defective in association with eithe r Skp1 or Cln2. Cln2 was highly stabilized and accumulated in the phosphory lated forms in the mutant cells. Furthermore, Skp1 associated in vivo with phosphorylated Cln2 in a Grr1-dependent manner. These data suggest that Grr 1 is required for degradation of Cln2 through linking phosphorylated Cln2 t o Skp1 in a SCFGrr1 complex.