Stimulating microtubule growth is not the essential function of the microtubule polymerase Stu2

ch-TOG family proteins, including the budding yeast Stu2, are essential for spindle formation and chromosome segregation. Such functions depend on an array of activities ranging from microtubule nucleation, polymerization and depolymerization, to conferring tension sensitivity to kinetochores. This functional diversity makes it challenging to dissect these various functions and understand their relative importance. Here, we developed separation-of-function mutants and used artificial tethering tools to elucidate several important mechanistic insights into Stu2s essential role. We show that Stu2s microtubule polymerization activity depends on its basic linker region but is surprisingly dispensable for viability; that in fact, Stu2 carries out an essential kinetochore-associated function; and finally, that Stu2s precise location within the kinetochore is critical for its function, suggesting a spatial separation mode of action may underlie its ability to confer tension sensitivity. Our findings highlight the significance of Stu2s kinetochore role and provide insights into the molecular mechanisms by which it performs its various functions.