Figure 1. Only one MELT motif per Spc105 is sufficient for SAC signaling in nocodazole-treated cells.
(A) A simplified schematic of the signaling cascade of the SAC in budding yeast (Mad3/BubR1, Cdc20, etc. are not shown). (B) Top: amino acid sequence alignment for the six MELT motifs in the Saccharomyces cerevisiae Spc105. Bottom: Crystal structure of the MELpT-Bub3-Bub1 complex (adapted from PDB 4BL0 and 2I3S). Note that the phosphothreonine (shown in blue, inter-chain hydrogen bonds highlighted by dashed lines) buried in a grove on Bub3 surface. (C) Micrographs display representative images of nocodazole-treated yeast cells with fluorescently labeled kinetochores and Bub1-mCherry. Asterisks mark Bub1-mCherry colocalized with the unattached kinetochore clusters. Kinetochores in the other, larger cluster proximal to the spindle pole bodies do not recruit SAC proteins, presumably because they remain attached to short microtubule stubs at the spindle pole bodies. Scale bar ~3.2 µm. Scatterplots show the quantification of Bub1-mCherry fluorescence in wild-type (WT) and mutant strains (labeled by the number designation of the active MELT motif). Fluorescence values have been normalized to the average Bub1-mCherry fluorescence in wild-type cells. (mean+ s.e.m. Top: n = 55, 33, 51 and 32 for WT, #2, #4 and #6 respectively, accumulated from two technical replicates. Bottom: n = 55, 92 and 68 for WT, #2 and #6 respectively, pooled from two technical replicates. n.s. Not significant. (D) Quantification of DNA content in cells treated with nocodazole using flow cytometry. The assay was performed once.
Figure 1—figure supplement 1. A The fraction of cells with 4N ploidy in the flow cytometry experiments shown in Figure 1D.
