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Biol Open. 2018 Oct 8;7(10). pii: bio033647. doi: 10.1242/bio.033647.

Novel phosphorylation states of the yeast spindle pole body.

Author information

1
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
2
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
3
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA tdavis@uw.edu.

Abstract

Phosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of enriched Saccharomyces cerevisiae SPBs 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 a cdc4-1 temperature-sensitive mutation, with particular focus on phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest 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 analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally, in vivo analysis 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 that influence microtubule formation.

KEYWORDS:

Cell cycle; Phosphoproteome; Spindle pole body (SPB); γ-tubulin small complex (γ-TuSC)

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