FIG. 3.

Analysis of Cln2 binding by Grr1 with alterations in the basic residues of the LRR. (A) Cln2 binding by Grr1 with basic residues of the LRR replaced by glutamine (Q). Anti-HA immunoprecipitates were prepared from extracts (WCE) of strains expressing wild-type or mutant GRR1-HA expressed from the ADH1 promoter and CLN2 expressed from the GAL1 promoter. Immunoblots were probed with the anti-HA or anti-Cln2 as indicated. Grr1-B4Q represents the protein with all four basic residues on the concave surface of the LRR converted to glutamine. (B) Interaction of Grr1 point mutants with glutamate replacement of basic residues with Cln2. This experiment was similar to that shown in panel A, except that the basic residues in the LRR of Grr1 were replaced by glutamate (E) instead of glutamine. Grr1-B4E represents the protein with all four basic residues on the concave surface of the LRR converted to glutamate.