Phospho-regulation of gamma-tubulin in budding yeast

Chromosome segregation is the crucial step for cell division in most living organisms. In order to segregate the chromosomes equally into mother and daughter cells, the cell needs to build a spindle, which comprises three major components: chromosomes, microtubules and microtubule organizing center (centrosome in animal cells or spindle pole body in fungal cells). The building and normal execution of spindle function are tightly linked to cell cycle control system.;γ-Tubulin is an evolutionally conserved protein with its canonical function as a microtubule nucleator. Here we report that γ-tubulin is phosphorylated at residue S360, a Cdk1/Cdc28 site. Phosphorylation of S360 in vivo was identified in a global analysis of the phosphoproteome of the spindle pole body (J. Keck and M. Jones, et al.). We confirmed Cdc28-Clb2 can phosphorylate S360 by in vitro kinase assay and peptide identification by mass spectrometry, and in vivo assay using two dimensional-polyacrylamide gel electrophoresis (2D-PAGE). A phospho-mimetic mutation (tub4-S360D) causes mitotic delay but does not inhibit recruitment of the γ-tubulin complex (reported by GRIP Spc97-EGFP) to spindle poles. Analysis of synthetic genetic interactions and live cell imaging revealed that cytoplasmic microtubule function is normal in tub4-S360D cells but spindle microtubule function is perturbed. High-resolution analysis of spindle dynamics revealed fluctuations in length in metaphase and anaphase spindles. The velocities of spindle elongation in anaphase are similar in S360D and WT spindles, but the initial phase of rapid elongation in anaphase is prolonged in the S360D mutant, and spindle breakdown is delayed.;Interpolar microtubules play a central role in spindle assembly and are actively responsible for spindle elongation with sliding forces generated from microtubule cross-linking proteins such as kinesin-5 (Cin8 in budding yeast). Genetic analysis indicates that mutations in proteins that act redundantly to Cin8 enhance the growth defect in S360D, and S360A can partially suppress deletion of Cin8. High-resolution tomography analysis of S360D mutant and WT cells reveals that the interpolar microtubules organization in S360D is completely perturbed, as almost none overlapping of interpolar microtubules were observed.;Therefore, we conclude that S360 phosphorylation of γ-tubulin/Tub4 by Cdk1/Cdc28 plays an important role in the control of spindle microtubule organization during spindle assembly, through docking and/or monitoring critical microtubule associated proteins, for example Cin8, at microtubule minus end hence regulating their functions on the plus end.