SUMMARY
Proteins that recognize and act on specific subsets of microtubules (MTs) enable the varied functions of the MT cytoskeleton. We recently discovered that Kif15 localizes exclusively to kinetochore-fibers (K-fibers) [1, 2], or bundles of kinetochore-MTs within the mitotic spindle. It is currently speculated that the MT-associated protein TPX2 loads Kif15 onto spindle MTs [3–5], but this model has not been rigorously tested. Here, we show that Kif15 accumulates on MT bundles as a consequence of two inherent biochemical properties. First, Kif15 is self-repressed by its C-terminus. Second, Kif15 harbors a non-motor MT-binding site, enabling dimeric Kif15 to crosslink and slide MTs. Two-MT-binding activates Kif15, resulting in its accumulation on and motility within MT bundles but not on individual MTs. We propose that Kif15 targets K-fibers via an intrinsic two-step mechanism involving molecular unfolding and two-MT-binding. This work challenges the current model of Kif15 regulation and provides the first account of a kinesin that specifically recognizes a higher order MT array.