Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast.
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.
Cuschieri
Lara
L
Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1.
Miller
Rita
R
Vogel
Jackie
J
D000199:Actins; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D008868:Microtubule Proteins / Q000378:metabolism* / Q000502:physiology; D008870:Microtubules / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D024701:Myosin Type V / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004718:Saccharomycetales / Q000378:metabolism; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000235:genetics / Q000502:physiology*
D000199:Actins; C119683:BIM1 protein, S cerevisiae; D018797:Cell Cycle Proteins; C110443:KAR9 protein, S cerevisiae; C077512:MYO2 protein, S cerevisiae; D008868:Microtubule Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C100764:TUB4 protein, S cerevisiae; D014404:Tubulin; D024701:Myosin Type V; D018995:Myosin Heavy Chains