Figure 8.
Point mutations within the SpCnp3CENP-C cupin domain exclusively affect its function in meiosis, through disruption of Moa1 recruitment to centromeres. A, SpCnp3CENP-C cupin domain point mutants (pocket and surface) largely display no TBZ or cold sensitivity when compared with cnp3Δ cells (note that moa1Δ cells also display no TBZ or cold sensitivity). 5-fold serial dilutions of cells of the indicated genotypes were spotted on YES medium supplemented with or without the indicated concentrations of TBZ and incubated at the indicated temperatures for 3–7 days. Two independent isolates of each genotype (cupin domain mutant) are shown. Point mutants unique to this study are in bold, whereas previously described mutants are not (23). B, point mutations within the SpCnp3CENP-C cupin pocket (F541A, A552T, and M574T) and surface (V566A, T586A, and Y607C) result in failure to recruit Moa1 to centromeres during meiosis I. Homothallic (h90) fission yeast cells of the indicated genotypes expressing GFP-tagged Moa1 were induced into meiosis at 32 °C and arrested in meiotic prophase I using a mei4Δ allele. DNA was stained with 4′,6′-diamino-2-phenylindole, following which cells were imaged live. Point mutants unique to this study are in bold, whereas previously described mutants are not (23). Scale bar, 10 μm. Also see Fig. S4. DIC, differential interference contrast.