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MBC in Press, published online ahead of print August 9, 2006
Mol. Biol. Cell 10.1091/mbc.E06-03-0245

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Submitted on March 28, 2006
Revised on July 27, 2006
Accepted on July 31, 2006

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

Lara Cuschieri,* Rita Miller,{dagger} and Jackie Vogel*

*Department of Biology, McGill University, Montreal, Québec, Canada H3A 1B1; {dagger}Department of Biology, University of Rochester, Rochester, NY 14627

Monitoring Editor: Kerry Bloom

Microtubule +end interacting proteins (+TIPs) promote the dynamic interactions between the +ends of astral microtubules and cortical actin that are required for pre-anaphase spindle positioning. Paradoxically, +TIPs such as the EB1 ortholog Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of 4 c-terminal residues of the budding yeast {gamma}-tubulin Tub4 (tub4-{Delta}dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependant 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 are suppressed in tub4-{Delta}dsyl cells, but are restored by deletion of Kar9. Moreover, Myo2 and F-actin dependant 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.

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




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