(A) ‘Spin-down’ assays for microtubule assembly reveal by SDS-PAGE that mutations on the lateral interface of β-tubulin (left, F281A) or α-tubulin (right, H284A) reduce the extent of microtubule assembly compared to wild-type. S = supernatant, p=pellet. Assays were performed using 1 μM tubulin. (B) A gel filtration binding assay demonstrate that a lateral tubulin mutant, Tub1-H284A, binds a TOG domain comparably to wild-type tubulin. Left: Overlaid gel filtration traces of WT tubulin alone (blue), TOG1 alone (red), and the tubulin + TOG mix (black). The shift to earlier elution volumes indicates formation of a larger complex. Right: Overlaid gel filtration traces of α-H284A tubulin alone (blue), TOG1 alone (red, repeated from left panel), and the tubulin + TOG mix (black). A similar shift in elution indicating the presence of a TOG:tubulin complex was observed. All samples contain 1 μM tubulin variant and 1 µM TOG1. (C) Analysis of TOG1:αβ-tubulin interactions by sedimentation velocity. The plot shows c(s) distributions (color coded by sample) for each control sample: TOG1 alone (2 µM, purple), WT tubulin (0.3 µM, blue), ‘LR1’ plus-end blocked tubulin (β-tubulin: T175R, V179R) (0.3 µM, red) and ‘F281A’ (β-tubulin: F281A) (0.3 µM, green). (D) Isotherms for Stu2 TOG1 domain binding to WT tubulin (black), plus-end blocked tubulin ‘LR1’ (β-tubulin:T175R,V179R) (blue) and side-block tubulin ‘F281A’ (β-tubulin:F281A) (red). TOG1 binds with comparable affinity to all three tubulin variants including the two tubulin mutants (plus-end and side-block) and WT tubulin. Fitted binding affinities and 1 σ confidence intervals for fitted affinities (in parentheses) are shown in the table on the right.