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. 2015 Jul 30;10(7):e0134297. doi: 10.1371/journal.pone.0134297

Fig 1. Lack of the Hop1-phospho-S298 leads to temperature- and dose- dependent meiotic failure.

Fig 1

(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 HOP1 and hop1-S298A panel (B) or HOP1 and hop1-T318A 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). In vivo phosphorylation of Hop1-S298 and T318 during DMC1 (D) or dmc1Δ (E) meiosis at 23°C. Nuclear spreads of a DMC1 or dmc1Δ 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, hop1-S298Ax2: an allele containing two tandem copies of hop1-S298A. hop1-SCD: an allele where the S298, S311, and T318 in the SCD are mutated to A [6]. hop1-S311A: an allele where the S311 is mutated to A. (G) Spore viability of indicated HOP1 alleles at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1). (H) Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background. (I). Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1).