FIGURE 6:

Consequences of mutating the tubulin-binding interface of Stu1-TOG2. (A) W339A (blue) or R525A (green) mutations on the tubulin-binding interface abolish anti-catastrophe (left) and rescue (right) activity comparably to the double mutant W339A, R525A (purple). R386A (pink), a mutation of the CLASP-specific residue identified from the structure, also abolishes anti-catastrophe and rescue-promoting activity. Assays were performed using yeast αβ-tubulin (0.8 µM) with mutant TOG2 (0.2 µM). Catastrophe events for a single trial per mutant are summarized as a survival plot with average frequency (in min⁻¹) and number of measured catastrophes as follows: W339A, 0.118, 153; R525A, 0.0775, 94; W339A, R525A, 0.082, 98; R386A, 0.103, 60. Data with (red) and without (gray) TOG2 are duplicated from Figure 5. (B) W339A (top) and R525A (middle) substantially weaken interactions with unpolymerized αβ-tubulin as detected by sedimentation velocity analytical ultracentrifugation; the double mutant W339A, R252A (bottom) and the CLASP-specific mutant R386A essentially abolishes interactions with tubulin. c(s) distributions are shown for tubulin only (black) and tubulin+mutant (green). (C) Results of a genetic rescue assay in which endogenous Stu1 can be degraded in an auxin-dependent manner, leading to a loss in cell viability. While plasmid-based expression of nondegradable wild-type, full-length Stu1 rescues the inducible growth defect, W339A, R529A, W339A, R529A (double mutant), and R386A point mutations in the full-length protein do not. STU1-AID cells expressing various covering alleles (as listed) were serially diluted and spotted on plates with either DMSO or auxin.