Figure 1.

Identification of Ndj1 as an SPB-associated protein. (A) Schematic diagram showing a pair of side-by-side SPBs during yeast meiosis. ONM, outer nuclear membrane; INM, inner nuclear membrane; OP, outer plaque; CP, central plaque; IP, inner plaque. (B) Comparison of SPB dynamics in vegetative and meiotic yeast cells. (C) A silver-staining gel showing the enrichment of SPB components after affinity purification of Spc97-TAP. Strain HY3674 was used. (D) List of SPB proteins identified by protein mass spectrometry of Spc97-TAP samples. Note that Ndj1 is meiosis specific. The extended list of peptides recovered by mass spectrometry of Spc97-TAP samples is available in Fig. S1. (E and F) Protein affinity purification of Ndj1-TAP (HY3813) and Mps3-TAP (HY3848) from meiotic yeast cells. Arrows point to the same protein bands identified by silver staining (left) and immunoblotting (right). Anti-GFP antibody was used to probe Mps3-GFP, and anti-HA antibody was used to probe Ndj1-3HA. Both antibodies also recognize Ndj1-TAP and Mps3-TAP. Representative proteins identified by protein mass spectrometry are listed in the tables below. (G) Localization of Ndj1 during meiosis. Yeast cells (HY3859) were induced to undergo meiosis, and time-lapse fluorescence microscopy was performed to localize Ndj1-GFP (green) and Spc42-RFP (red). Projected images of eight z sections are shown. Time zero is defined as the point of SPB separation. Arrows point to the Ndj1-GFP focus at the SPB. The graph below shows the relative intensity of the Ndj1-GFP focus at the SPB before SPB separation. The representative cell shown is from a single time-lapse experiment (n > 50). The SPB area was defined by the Spc42-RFP signal. Bar, 2 µm. (H) A timeline of Ndj1 localization, disassociation, and SPB separation during meiosis I. Duration of Ndj1 localization at SPB is not drawn to scale.