Cdk1 phosphorylation inhibits the interaction of Exo84 with other exocyst components. (A) Immunoblots of cells expressing wild-type and mutant Exo84 (Exo84-A and Exo84-E) proteins. Exo84 was detected using a rabbit anti-Exo84 antibody. ADH was probed as a protein loading control. (B) Exo84 phosphorylation negatively regulates exocyst assembly. Cells were grown to early log phase at 25°C. Then wild-type and mutant Exo84 were immunoprecipitated from cells coexpressing a GFP-tagged exocyst protein (Sec5, Sec10, Sec15, or Exo70, tagged by chromosomal integration). Coimmunoprecipitated exocyst proteins were probed with an anti-GFP antibody. The relative amounts of coprecipitated Sec10-GFP, Sec15-GFP, and Exo70-GFP were higher in the exo84-A mutant and lower in the exo84-E mutant. The amount of coimmunoprecipitated Sec5-GFP is similar for all Exo84 variants. The quantification of the binding is shown below each Western blot. Error bars represent standard deviation. *, P < 0.01; n = 3. (C) Phosphorylation negatively regulates Exo84 interaction with Sec10. Exo84, Exo84-A, and Exo84-E were synthesized by in vitro transcription/translation in the presence of [³⁵S]methionine/cysteine. The proteins were then incubated with purified recombinant His₆-tagged Sec10 conjugated to Ni-Sepharose. After extensive washing, the bound proteins were analyzed by SDS-PAGE and autoradiography. The Coomassie blue–stained gel (top) shows the inputs of His₆-tagged Sec10 protein. The autoradiograph (bottom) shows the input (10%) and bound [³⁵S]methionine-labeled Exo84 and Exo84 phospho mutants (Exo84-ps). The binding of His₆-Sec10 with Exo84 and exo84 was quantified in D. Error bars represent standard deviation (n = 3). *, P < 0.01.