4.5 Article

Measuring Affinities of Fission Yeast Spindle Pole Body Proteins in Live Cells across the Cell Cycle

Journal

BIOPHYSICAL JOURNAL
Volume 105, Issue 6, Pages 1324-1335

Publisher

CELL PRESS
DOI: 10.1016/j.bpj.2013.08.017

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Funding

  1. National Institutes of Health [GM026132]
  2. National Science Foundation Predoctoral Fellowship

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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 K-d < 0.1 mu M when Sid2p binds Mob1p-Cdc1lp and Kd in the micromolar range when Mob1 p 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 Cdcl1p at SPBs with lower affinity: K(d)s > 10 mu M 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.

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