F-box protein Grr1 interacts with phosphorylated targets via the cationic surface of its leucine-rich repeat

The flexibility and specificity of ubiquitin-dependent proteolysis are medi ated, in part, by the E3 ubiquitin ligases. One class of E3 enzymes, SKp1/c ullin/F-box protein (SCF), derives its specificity from F-box proteins, a h eterogeneous family of adapters for target protein recognition. Grr1, the F -box component of SCFGrr1 mediates the interaction with phosphorylated form s of the G(1) cyclins Cln1 and Cln2. We show that binding of Cln2 by SCFGrr 1 was dependent upon its leucine-rich repeat (LRR) domain and its carboxy t erminus. Our structural model for the Grr1 LRR predicted a high density of positive charge on the concave surface of the characteristic horseshoe stru cture. We hypothesized that specific basic residues on the predicted concav e surface are important for recognition of phosphorylated Cln2. We show tha t point mutations that converted the basic residues on the concave surface but not those on the convex surface to neutral or acidic residues interfere d with the capacity of Grr1 to bind to Cln2. The same mutations resulted in the stabilization of Cln2 and Gic2 and also in a spectrum of phenotypes ch aracteristic of inactivation of GRR1, including hyperpolarization and enhan cement of pseudohyphal growth. It was surprising that the same residues wer e not important for the role of Grr1 in nutrient-regulated transcription of HXT1 or AGP1, We concluded that the cationic nature of the concave surface of the Grr1 LRR is critical for the recognition of phosphorylated targets of SCFGrr1 but that other properties of Grr1 are required for its other fun ctions.