Lack of the Hop1-phospho-S298 leads to temperature- and dose- dependent meiotic failure.

<p>(A) Schematic representation of Hop1 with the locations of eight [S/T]Q motifs. S: serine, T: threonine, SCD: [S/T]Q Cluster Domain. Also shown are the HORMA domain, Zn finger motif, and nuclear localization signal (NLS). (B) and (C) Specificity of the phospho-specific α-pS298 and α-pT318 antibodies. Nuclear spreads of <i>HOP1</i> and <i>hop1-S298A</i> panel (B) or <i>HOP1</i> and <i>hop1-T318A</i> panel (C) were prepared from samples taken at 5hours after induction of synchronous meiosis at 23°C. The spreads were stained with DAPI and the antibodies against either the phospho-S298 panel (B) or the phospho-T318 panel (C). (D) and (E). <i>In vivo</i> phosphorylation of Hop1-S298 and T318 during <i>DMC1</i> (D) or <i>dmc1Δ</i> (E) meiosis at 23°C. Nuclear spreads of a <i>DMC1</i> or <i>dmc1Δ</i> strain were prepared from samples collected at the indicated time points. The spreads were stained with the antibodies against Hop1, HA (for detection of Mek1-HA), and the two phospho-specific antibodies, α-pS298 and α-pT318. A nucleus exhibiting 10 or more foci of each epitope was scored as a positive. Also shown are the fractions of cells having undergone first meiotic division or meiosis I (MI) at each time point. Errors were calculated from the 95% confidence interval of a binomial distribution. (F) Spore viability of homozygous diploid strains of indicated genotype at specified temperature. For each genotype, at least 80 spores were analysed. A: Alanine; D: aspartic acid, <i>hop1-S298Ax2</i>: an allele containing two tandem copies of <i>hop1-S298A</i>. <i>hop1-SCD</i>: an allele where the S298, S311, and T318 in the SCD are mutated to A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134297#pone.0134297.ref006" target="_blank">6</a>]. <i>hop1-S311A</i>: an allele where the S311 is mutated to A. (G) Spore viability of indicated <i>HOP1</i> alleles at 23°C as a either homozygous diploid (<i>allele/allele</i>), hemizygous diploid (<i>allele</i>/ <i>hop1Δ</i>) or heterozygous diploid (<i>allele/HOP1</i>). (H) Sporulation efficiency of <i>dmc1Δ</i> strains in the indicated <i>hop1</i> mutation background. (I). Sporulation efficiency of <i>dmc1Δ</i> strains in the indicated <i>hop1</i> mutation background at 23°C as a either homozygous diploid (<i>allele/allele</i>), hemizygous diploid (<i>allele</i>/ <i>hop1Δ</i>) or heterozygous diploid (<i>allele/HOP1</i>).