title: Unraveling interactions of cell cycle-regulating proteins Sic1 and B-type cyclins in living yeast cells: a FLIM-FRET approach.
abstract: Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.
authors: Theoretical Biophysics, Department of Biology, Humboldt Universität zu Berlin, Berlin, Germany.
authors: Gabriele
authors: G
authors: Schreiber
authors: Matteo
authors: M
authors: Barberis
authors: Silvia
authors: S
authors: Scolari
authors: Christine
authors: C
authors: Klaus
authors: Andreas
authors: A
authors: Herrmann
authors: Edda
authors: E
authors: Klipp
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C054562:yellow fluorescent protein, Bacteria; D016203:CDC2 Protein Kinase