Identification of Putative Mek1 Substrates during Meiosis in <i>Saccharomyces cerevisiae</i> Using Quantitative Phosphoproteomics

Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast.减数分裂重组（Meiotic recombination）在有性生殖中发挥关键作用：它可产生交叉互换（crossovers），并与姐妹染色单体黏连（sister chromatid cohesion）共同作用，在物理层面连接同源染色体（homologous chromosomes），进而促进同源染色体在第一次减数分裂时正确分离。减数分裂重组起始于程序性双链断裂（double strand breaks, DSBs），该断裂由进化保守的拓扑异构酶样蛋白Spo11催化完成。此类双链断裂的修复受到严格调控，以在分布于整个基因组的同源染色体之间形成交叉互换。该修复过程需要有丝分裂重组酶Rad51的参与，同时也依赖于减数分裂特异性重组酶Dmc1的链交换活性。在酿酒酵母（Saccharomyces cerevisiae）中，减数分裂双链断裂修复的关键调控因子是减数分裂特异性激酶Mek1：它可促进同源链入侵，同时是减数分裂重组检验点以及交换/非交换命运决定所必需的。解析Mek1调控减数分裂重组的机制，需要确定其底物蛋白。为此，研究人员采用了基于细胞内氨基酸稳定同位素标记（Stable Isotope Labeling by Amino Acids in Cells, SILAC）的无偏倚磷酸蛋白质组学方法，筛选得到了一系列潜在的Mek1底物蛋白，以及携带细胞周期蛋白依赖性激酶、检验点激酶Mec1/Tel1和polo样激酶Cdc5共有磷酸化位点的蛋白。本实验首次构建了减数分裂酿酒酵母蛋白的全局磷酸蛋白质组数据集。