TORC1 kinase and the S-phase cyclin Clb5 collaborate to promote mitotic spindle assembly and DNA replication in S. cerevisiae
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The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild-type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression.
KeywordsRapamycin Kip3 Clb5 Cell cycle S phase Microtubules
This work was funded by NSF Grant MCB-0416738 and a Staley Fellowship for Cancer-Related Research at Wellesley College to J.K.H. and by NIH GM07663 to D.M.K. The authors thank Jennifer Ferguson, Langdon Smythe, and Katie Montelione for technical assistance and Fred Cross, Andy Hoyt, Yoshi Kamada, and David Pellman for generously sharing plasmids and yeast strains.
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