FIG 5.
PKA phosphorylation and GAP activity inactivation of Gyp1 enhance its association with Myo2. (A) Log-phase yeast cells coexpressing Myo2-hemagglutinin (Myo2-HA) with Gyp1 (HZX232), Gyp1-GFP (HZX228), Gyp14A-GFP (HZX230), Gyp14E-GFP (HZX231), or Gyp1R292K-GFP (HZX229) were subjected to immunoprecipitation (IP) with anti-HA antibody to pull down Myo1, and the precipitation products were analyzed by Western blotting using anti-GFP and anti-HA antibody. The band intensity of the GFP-tagged Gyp1 protein was normalized to the band intensity of Myo2-HA in the same sample (lower panel). The experiment was done three times, and the differences between WT Gyp1 and mutant Gyp1 proteins were statistically significant (*, P < 0.05; **, P < 0.01 [t test]). Error bars represent SEM. (B and C) Localization of Gyp1R292K-GFP (HZX203) and Gyp14A,R292K-GFP (HZX233) in yeast and hyphal cells.