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-box proteins, have been implicated in Cdc34-dependent ubiquitination. Grr1, which is required for degradation of G1 cyclins Cln1 and Cln2 as well as for regulation of glucose repression, is an F-box protein and interacts with Skp1 through the F-box motif. Grr1 also interacts in vitro with phosphorylated Cln1 and Cln2. However, ubiquitination of Cln1 has not been successful 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 has 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 Grr1 with Skp1 but not with phosphorylated Cln2 in vivo. Based on these results, we constructed grr1 mutants that are defective in association with either Skp1 or Cln2. Cln2 was highly stabilized and accumulated in the phosphorylated forms in the mutant cells. Furthermore, Skp1 associated in vivo with phosphorylated Cln2 in a Grr1-dependent manner. These data suggest that Grr1 is required for degradation of Cln2 through linking phosphorylated Cln2 to Skp1 in a SCFGrr1 complex.
Kishi
T
T
Laboratory of Mutagenesis, Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan. tkishi@lab.nig.ac.jp.
Yamao
F
F
D002352:Carrier Proteins / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000502:physiology*; D007930:Leucine / Q000378:metabolism; D008025:Ligases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D020449:Repetitive Sequences, Amino Acid; D044786:S-Phase Kinase-Associated Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases
C082721:CLN2 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D044783:F-Box Proteins; D005656:Fungal Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; C071032:GRR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D007930:Leucine