Phosphosites of the yeast centrosome component Spc110 contribute to cell cycle progression and mitotic exit

ABSTRACT Spc110 is an essential component of the spindle pole body (SPB), the yeast equivalent of the centrosome, that recruits the γ-tubulin complex to the nuclear side of the SPB to produce the microtubules that form the mitotic spindle. Here, we identified phosphosites S11 and S36 in maternally originated Spc110 and explored their functions in vivo. Yeast expressing non-phosphorylatable Spc110S11A had a distinct spindle phenotype characterised by higher levels of α-tubulin, which was frequently asymmetrically distributed between the two SPBs. Furthermore, expression of the double mutant Spc110S11AS36A had a delayed cell cycle progression. Specifically, the final steps of mitosis were delayed in Spc110S11AS36A cells, including expression and degradation of the mitotic cyclin Clb2, disassembling the mitotic spindle and re-localizing Cdc14 to the nucleoli, resulting in late mitotic exit and entry in G1. Thus, we propose that Spc110 phosphorylation at S11 and S36 is required to regulate timely cell cycle progression in budding yeast. This article has an associated First Person interview with the first author of the paper.

[1]  F. Monje-Casas,et al.  Polo-like kinase acts as a molecular timer that safeguards the asymmetric fate of spindle microtubule-organizing centers , 2020, eLife.

[2]  Y. Arava,et al.  Expanding the CRISPR/Cas9 Toolbox for Gene Engineering in S. cerevisiae , 2020, Current Microbiology.

[3]  F. Monje-Casas,et al.  Asymmetric inheritance of spindle microtubule-organizing centres preserves replicative lifespan , 2019, Nature Cell Biology.

[4]  Ethan J. Sanford,et al.  In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome , 2019, bioRxiv.

[5]  G. Rancati,et al.  Recruitment of the mitotic exit network to yeast centrosomes couples septin displacement to actomyosin constriction , 2018, Nature Communications.

[6]  Kimberly K. Fong,et al.  Novel phosphorylation states of the yeast spindle pole body , 2018, Biology Open.

[7]  Ann M. Cavanaugh,et al.  Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function. , 2017, Annual review of genetics.

[8]  K. Baetz,et al.  Budding yeast Wee1 distinguishes spindle pole bodies to guide their pattern of age-dependent segregation , 2017, Nature Cell Biology.

[9]  E. Queralt,et al.  Regulation of Mitotic Exit in Saccharomyces cerevisiae. , 2017, Methods in molecular biology.

[10]  Wei-Lih Lee,et al.  Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae , 2015, Traffic.

[11]  S. Jaspersen,et al.  Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1 , 2014, PLoS genetics.

[12]  E. Nigg,et al.  Centrosomes as signalling centres , 2014, Philosophical Transactions of the Royal Society B: Biological Sciences.

[13]  E. Schiebel,et al.  Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle , 2014, Current Biology.

[14]  Lisa Weber,et al.  Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation , 2014, eLife.

[15]  Michael B. Yaffe,et al.  Activation of the Yeast Hippo Pathway by Phosphorylation-Dependent Assembly of Signaling Complexes , 2013, Science.

[16]  J. Neefjes,et al.  Spatiotemporal analysis of organelle and macromolecular complex inheritance , 2012, Proceedings of the National Academy of Sciences.

[17]  Daici Chen,et al.  Spindle Pole Bodies Exploit the Mitotic Exit Network in Metaphase to Drive Their Age-Dependent Segregation , 2012, Cell.

[18]  A. Amon,et al.  Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit. , 2011, Genes & development.

[19]  Jill Falk,et al.  Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4 , 2011, Proceedings of the National Academy of Sciences.

[20]  Arend Sidow,et al.  A Cell Cycle Phosphoproteome of the Yeast Centrosome , 2011, Science.

[21]  G. Pereira,et al.  The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4 , 2011, The Journal of cell biology.

[22]  E. Schiebel,et al.  Phosphorylation of the Yeast γ-Tubulin Tub4 Regulates Microtubule Function , 2011, PloS one.

[23]  F. Monje-Casas,et al.  Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function , 2011, The Journal of cell biology.

[24]  Hongwei Wang.  Faculty Opinions recommendation of Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry. , 2010 .

[25]  A. Ciliberto,et al.  Oscillations in Cdc14 release and sequestration reveal a circuit underlying mitotic exit , 2010, The Journal of cell biology.

[26]  H. Ovaa,et al.  Recombination-induced tag exchange to track old and new proteins , 2009, Proceedings of the National Academy of Sciences.

[27]  D. Gottschling,et al.  The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae , 2009, Genetics.

[28]  Raymond J. Deshaies,et al.  Dbf2–Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis , 2009, The Journal of cell biology.

[29]  Angelika Amon,et al.  Mitotic CDKs control the metaphase-anaphase transition and trigger spindle elongation. , 2008, Genes & development.

[30]  S. M. Huisman,et al.  Phosphorylation of Spc110p by Cdc28p-Clb5p kinase contributes to correct spindle morphogenesis in S. cerevisiae , 2007, Journal of Cell Science.

[31]  E. Salmon,et al.  The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit. , 2004, Molecular biology of the cell.

[32]  T. Davis,et al.  The Saccharomyces cerevisiae spindle pole body is a dynamic structure. , 2003, Molecular biology of the cell.

[33]  S. Yoshida,et al.  Mitotic Exit Network Controls the Localization of Cdc14 to the Spindle Pole Body in Saccharomyces cerevisiae , 2002, Current Biology.

[34]  Angelika Amon,et al.  Separase, Polo Kinase, the Kinetochore Protein Slk19, and Spo12 Function in a Network that Controls Cdc14 Localization during Early Anaphase , 2002, Cell.

[35]  K. Nasmyth,et al.  Modes of spindle pole body inheritance and segregation of the Bfa1p–Bub2p checkpoint protein complex , 2001, The EMBO journal.

[36]  M. Snyder,et al.  Phosphorylation of γ-Tubulin Regulates Microtubule Organization in Budding Yeast , 2001 .

[37]  A. Amon,et al.  Regulation of the mitotic exit protein kinases Cdc15 and Dbf2. , 2001, Molecular biology of the cell.

[38]  R. Deshaies,et al.  Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  T. Davis,et al.  Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain* , 2001, The Journal of Biological Chemistry.

[40]  A. Amon,et al.  A Mechanism for Coupling Exit from Mitosis to Partitioning of the Nucleus , 2000, Cell.

[41]  J. Grindlay,et al.  The Bub2p spindle checkpoint links nuclear migration with mitotic exit. , 2000, Molecular cell.

[42]  G. Braus,et al.  Two different modes of cyclin Clb2 proteolysis during mitosis in Saccharomyces cerevisiae , 2000, FEBS letters.

[43]  M. Knop,et al.  Interaction of the yeast γ‐tubulin complex‐binding protein Spc72p with Kar1p is essential for microtubule function during karyogamy , 1999, The EMBO journal.

[44]  Angelika Amon,et al.  Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus , 1999, Nature.

[45]  Anna Shevchenko,et al.  Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex , 1999, Cell.

[46]  M. Knop,et al.  Receptors determine the cellular localization of a γ‐tubulin complex and thereby the site of microtubule formation , 1998, The EMBO journal.

[47]  M. Knop,et al.  Spc98p and Spc97p of the yeast γ‐tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p , 1997, The EMBO journal.

[48]  M. Werner-Washburne,et al.  Stationary phase in Saccharomyces cerevisiae , 1996, Molecular microbiology.

[49]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.