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Vol. 17, Issue 10, 4420-4434, October 2006

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

Lara Cuschieri^*, Rita Miller, and Jackie Vogel^*

*Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1; and Department of Biology, University of Rochester, Rochester, NY 14627

Submitted March 28, 2006; Revised July 27, 2006; Accepted July 31, 2006
Monitoring Editor: Kerry Bloom

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 -tubulin Tub4 (tub4-dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependant spindle positioning. Surprisingly, we find Kar9 localizes to microtubule +ends in tub4-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-dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-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-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-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.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-03-0245) on August 9, 2006.

Address correspondence to: Jackie Vogel (jackie.vogel{at}mcgill.ca)

Abbreviations used: +end, microtubule plus end; +TIP, plus tip protein; SPB, spindle pole body.

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