Microtubule binding by WT GCN4-Kar3Vik1. MT•Kar3Vik1 cosedimentation was performed to evaluate the binding properties based on the nucleotide state (A–F). 1 mM MgADP (A and B), 100 µM MgADP followed by apyrase treatment (C and D), and 1 mM MgAMPPNP (E and F). The supernatant (S) and pellet (P) for each reaction are shown with the gels, and the concentration of tubulin polymer is identified above each pair. The fraction of 2 µM WT GCN4-Kar3Vik1 that partitioned with MTs was plotted as a function of microtubule concentration. The fit of the data provided K_d,MT = 0.37 ± 0.07 µM for ADP (B), n = 3; K_d,MT = 0.36 ± 0.07 µM for nucleotide-free (D), n = 2; and K_d,MT = 0.04 ± 0.01 µM for AMPPNP (F), n = 3, with all three at ∼100% maximal binding. The stoichiometry of WT GCN4-Kar3Vik1 binding to microtubules was 1:2 for ADP (B) and nucleotide-free states (D) (i.e., 2 µM Kar3Vik1 per 4 µM αβ-tubulin), and 1:1 for AMPPNP (F; i.e., 2 µM Kar3Vik1 per 2 µM αβ-tubulin). Numbers to the right of the gels indicate the experimentally derived molecular masses (in kilodaltons) of the proteins being analyzed. No molecular weight markers were run on these gels.