Data_Sheet_1_Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast.PDF

The coordination of metabolism and growth with cell division is crucial for proliferation. While it has long been known that cell metabolism regulates the cell division cycle, it is becoming increasingly clear that the cell division cycle also regulates metabolism. In budding yeast, we previously showed that over half of all measured metabolites change concentration through the cell cycle indicating that metabolic fluxes are extensively regulated during cell cycle progression. However, how this regulation is achieved still remains poorly understood. Since both the cell cycle and metabolism are regulated to a large extent by protein phosphorylation, we here decided to measure the phosphoproteome through the budding yeast cell cycle. Specifically, we chose a cell cycle synchronization strategy that avoids stress and nutrient-related perturbations of metabolism, and we grew the yeast on ethanol minimal medium to force cells to utilize their full biosynthetic repertoire. Using a tandem-mass-tagging approach, we found over 200 sites on metabolic enzymes and transporters to be phospho-regulated. These sites were distributed among many pathways including carbohydrate catabolism, lipid metabolism, and amino acid synthesis and therefore likely contribute to changing metabolic fluxes through the cell cycle. Among all one thousand sites whose phosphorylation increases through the cell cycle, the CDK consensus motif and an arginine-directed motif were highly enriched. This arginine-directed R-R-x-S motif is associated with protein-kinase A, which regulates metabolism and promotes growth. Finally, we also found over one thousand sites that are dephosphorylated through the G1/S transition. We speculate that the phosphatase Glc7/PP1, known to regulate both the cell cycle and carbon metabolism, may play an important role because its regulatory subunits are phospho-regulated in our data. In summary, our results identify extensive cell cycle dependent phosphorylation and dephosphorylation of metabolic enzymes and suggest multiple mechanisms through which the cell division cycle regulates metabolic signaling pathways to temporally coordinate biosynthesis with distinct phases of the cell division cycle.

细胞代谢与生长与细胞分裂的协调对于增殖至关重要。尽管长期以来已知细胞代谢调节细胞分裂周期，但越来越明确的是，细胞分裂周期同样调节代谢。在酵母芽殖过程中，我们先前研究表明，所有测量的代谢物中超过一半在细胞周期中发生浓度变化，这表明代谢通量在细胞周期进程中受到广泛调控。然而，这种调控机制仍不甚明了。鉴于细胞周期和代谢在很大程度上都受蛋白质磷酸化所调控，我们在此决定通过酵母芽殖细胞周期测量磷酸蛋白组。具体而言，我们选择了一种避免代谢压力和营养相关扰动的细胞周期同步策略，并在乙醇最小培养基上培养酵母，以迫使细胞充分利用其全部生物合成库。采用串联质谱标记法，我们发现超过200个代谢酶和转运蛋白位点受到磷酸化调控。这些位点分布于众多途径中，包括碳水化合物分解、脂质代谢和氨基酸合成，因此可能通过细胞周期改变代谢通量。在所有细胞周期中磷酸化增加的一千个位点中，CDK共轭基序和精氨酸导向基序高度富集。这种精氨酸导向的R-R-x-S基序与蛋白激酶A相关，后者调节代谢并促进生长。最后，我们还发现超过一千个在G1/S转换过程中去磷酸化的位点。我们推测，已知可调节细胞周期和碳代谢的磷酸酶Glc7/PP1可能发挥重要作用，因为其调节亚基在我们数据中受到磷酸化调控。总之，我们的研究结果揭示了代谢酶在细胞周期中的广泛磷酸化和去磷酸化，并提出了多种机制，通过这些机制，细胞分裂周期调节代谢信号通路，以时空协调生物合成与细胞分裂周期的不同阶段。