Novel Phosphorylation States of the Yeast Spindle Pole Body

Abstract

ABSTRACTPhosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purifiedSaccharomyces cerevisiaeSPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by acdc4-1temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). Thecdc4-1arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzedin vivoby fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally,in vivoanalysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites influencing microtubule regulation.