Figure 3. Inactivating tubulin binding by the basic-proximal TOG domain enhances processivity of Stu2.

(A) Example raw fluorescence intensity trace showing two-step photobleaching of a stationary, homodimeric Stu2-eGFP. (B) Lifetime distribution for single Stu2-eGFP molecules on the growing microtubule end under single molecule conditions (5 nM Stu2, black) or ‘spiked’ into a higher concentration reaction (200 nM Stu2 of which only 5 nM is labeled, orange). Inset kymograph illustrates instances of Stu2 ‘runs’ (arrows). Histogram summarizes n = 400 measurements (four independent trials at 1 μM tubulin) for 5 nM Stu2-eGFP and n = 200 measurements (two independent trials at 0.8 μM tubulin) made using 5 nM Stu2-eGFP with 195 nM Stu2-KCK unlabeled. Data, which are plotted as percent of total for comparative purposes, were fit with an exponential, yielding an average residence time of 2.2 s (see also D) for single molecule eGFP and 2.0 s (see also D) for the spike measurements. Scale bar is 4 s. (C) Lifetime histograms for Stu2 variants. Compromising tubulin binding by the basic-proximal TOG (TOG1-TOG2*, red trace) yields a roughly 2-fold increase in end residence time. Compromising the N-terminal TOG (TOG1*-TOG2, blue trace) does not increase end-residence time. TOG2-TOG2* (green trace) also shows an increase in end residence time. All samples contained 5 nM Stu2-eGFP variants and 1 μM unlabeled yeast tubulin. Two independent trials of n = 100 measurements for all mutants, yielding a total n = 200. Samples were fit with exponential as done in B to extract average residence times. (D) Tabulated summary of results from all exponential fits to residence time distributions in C.