Figure 4. Polymerase elements required for preferential plus-end localization of Stu2.

Polymerase assays were carried out using wild-type tubulin (0.8 μM) and a panel of polymerase mutants (200 nM each). Representative kymographs are shown for Stu2 variants. Plus-end localization requires at least two tubulin-binding TOGs (WT, TOG1*-TOG2, ∆cc-2xBasic) and is not sensitive to how they are linked (compare TOG1*-TOG2 to ∆cc-2xBasic). Unexpectedly, polymerases with 0 (TOG1*-TOG2*) or one active TOGs (TOG1*-TOG2-∆cc-2xBasic) robustly coat the body of the microtubule. See also Figure 4—figure supplement 1.

Figure 4—figure supplement 1. Additional data from assays using mutated TOGs.

‘Double dead’ Stu2 (TOG1*-TOG2*) (left) with both TOGs inactivated does not show polymerase activity (red). Data for wild-type Stu2 (black) are reproduced from Figure 1. Stu2(TOG1*-TOG2)-∆cc-2xBasic (right), which contains a single active TOG domain, does not show polymerase activity (purple). Data for Stu2(TOG1*-TOG2)-∆cc-2xBasic (black; this construct has two active TOGs) are reproduced from Figure 1. Black: N = 45 from three independent chambers; Red and Purple: N = 20 measurements. All error bars are SEM. Smooth curves indicate a hyperbolic fit to the data.