Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.中隔蛋白（septins）是一类保守的GTP结合蛋白家族，在酿酒酵母中，它们可在母芽颈处组装为高度有序的细丝阵列。这类细丝在细胞周期进程中会发生显著的结构重排。本研究旨在鉴定参与或调控中隔蛋白动态转变的关键组分。通过结合细胞同步化技术与定量亲和纯化质谱（quantitative affinity-purification mass-spectrometry），我们针对细胞周期三个不同阶段的中隔蛋白特异性互作伴侣开展了筛选，最终共鉴定得到83个中隔蛋白的互作靶点。令人意外的是，我们在α因子阻滞、未形成组装态中隔蛋白结构的细胞中，主要检测到了DNA结合/核蛋白以及参与核糖体生物发生与蛋白质合成的蛋白。此外，本研究还鉴定到两类截然不同的调控蛋白组：一类特异性对应处于S期且具有稳定中隔蛋白环带的细胞，另一类则对应处于有丝分裂期且中隔蛋白环发生分裂的细胞。通过SPLIFF、免疫沉淀等互补实验方法，我们进一步明确了本次定量亲和纯化质谱筛选所得的部分候选靶点的时空分布特征。