Figure 3.

Identification of Mss4-interacting proteins by chemical crosslinking experiments and quantitative mass spectrometry. (A) Outline of the quantitative SILAC–MS approach. See the Results and Materials and methods for additional details. (B) Expression ratio (Xpress ratio=Mss4–3xFlag IP/ control IP) for proteins identified by the SILAC–MS experiments. An Xpress ratio of >10 was used as a set point to define specific Mss4-interacting proteins. Opy1 was enriched >50-fold in the Mss4–3xFlag IP compared with control IP. The inset shows the number of Mss4 and Opy1 peptides identified in the heavy (containing Mss4–3xFlag) and light samples. (C) Opy1–3HA crosslinks and coimmunoprecipitates with Mss4–Flag. Lysates from cells expressing Opy1–3HA or Mss4–Flag and Opy1–3HA were incubated with crosslinker and incubated with anti-Flag beads. Immunoprecipitates were analysed by immunoblotting to detect Mss4–Opy1 interactions. (D) Opy1 localizes to cortical structures and the cytoplasm. Wild-type cells expressing Opy1–GFP were grown to mid-log and examined by fluorescence microscopy. Cells shown are representative of over 100 cells observed. Scale bar, 4 μm. Diagram of the Opy1 protein is shown under the fluorescence images. PH, pleckstrin homology domain. Figure source data can be found with the Supplementary data.