γ-Tubulin Is Required for Proper Recruitment and Assembly of Kar9–Bim1 Complexes in Budding Yeast

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Abstract
Microtubule plus-end-interacting proteins (+TIPs) promote the dynamic interactions between the plus ends (+ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, +TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast gamma-tubulin Tub4 (tub4-delta dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependent spindle positioning. Surprisingly, we find Kar9 localizes to microtubule +ends in tub4-delta dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-delta dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-delta dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2- and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-delta dsyl cells, suggesting defective Kar9 complexes tether microtubule +ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-delta dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional +TIP complexes before their deployment to microtubule +ends.