Microtubule binding by nucleotide-free WT GCN4-Kar3Vik1. Kar3Vik1 was treated with apyrase to generate nucleotide-free Kar3Vik1 and then mixed with microtubules. The resulting MT•Kar3Vik1 complex was evaluated by sedimentation to determine the binding properties of this intermediate. (A) The supernatant (S) and pellet (P) for each reaction are shown with the gel, and the concentration of tubulin polymer is identified above each pair. (B) The fraction of 2 µM apyrase-treated Kar3Vik1 that partitioned with microtubules was plotted as a function of microtubule concentration. The data shown are from a single representative experiment. The fit of the data provided the apparent K_d,MT for each experiment with the mean ± SEM, K_d,MT = 0.003 ± 0.004 µM, n = 2. The stoichiometry of binding is somewhat less than 1:1; i.e., 2 µM Kar3Vik1 binds to ∼1.5 µM αβ-tubulin. Numbers to the right of the gels indicate the experimentally derived molecular weights (in kilodaltons) of the proteins being analyzed. No molecular weight markers were run on these gels.