title: Measuring affinities of fission yeast spindle pole body proteins in live cells across the cell cycle.
abstract: Characterizing protein-protein interactions is essential for understanding molecular mechanisms, although reproducing cellular conditions in vitro is challenging and some proteins are difficult to purify. We developed a method to measure binding to cellular structures using fission yeast cells as reaction vessels. We varied the concentrations of Sid2p and Mob1p (proteins of the septation initiation network) and measured their binding to spindle pole bodies (SPBs), the centrosome equivalent of yeast. From our measurements we infer that Sid2p and Mob1p both exist as monomeric, heterodimeric, and homodimeric species throughout the cell cycle. During interphase these species have widely different affinities for their common receptor Cdc11p on the SPB. The data support a model with a subset of Cdc11p binding the heterodimeric species with a Kd < 0.1 μM when Sid2p binds Mob1p-Cdc11p and Kd in the micromolar range when Mob1p binds Sid2p-Cdc11p. During mitosis an additional species presumed to be the phosphorylated Sid2p-Mob1p heterodimer binds SPBs with a lower affinity. Homodimers of Sid2p or Mob1p bind to the rest of Cdc11p at SPBs with lower affinity: Kds > 10 μM during interphase and somewhat stronger during mitosis. These measurements allowed us to account for the fluctuations in Sid2p binding to SPBs throughout the cell cycle.
authors.affiliation: Departments of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
authors.forename: Chad D
authors.initials: CD
authors.lastname: McCormick
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Akamatsu
authors.forename: Shih-Chieh
authors.initials: SC
authors.lastname: Ti
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pollard
mesh_terms: D002453:Cell Cycle; D002460:Cell Line; D007091:Image Processing, Computer-Assisted; D007399:Interphase; D008024:Ligands; D050296:Microbial Viability; D008856:Microscopy, Fluorescence / Q000379:methods*; D008938:Mitosis; D015388:Organelles / Q000378:metabolism; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000166:cytology*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D064048:Spindle Pole Bodies / Q000378:metabolism*
chemical_list: D008024:Ligands; D029702:Schizosaccharomyces pombe Proteins
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