The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.

中央代谢调节因子 SnRK1 在能量消耗激活后控制植物生长和存活，但对其调节和下游目标的详细分子洞察是有限的。在这里，我们使用磷酸蛋白质组学来推断以激酶饥饿为目标的蔗糖依赖性过程，如 SnRK1，证实了 SnRK1 与代谢酶和转录调节因子的关系，同时也指出了 SnRK1 对细胞内运输的控制。接下来，我们整合了亲和纯化、邻近标记和交联质谱法来绘制 SnRK1 异源三聚体的蛋白质相互作用图谱、组成和结构图，从而深入了解其植物特异性调控。在这个多维交互组的交汇处，我们发现 SnRK1 与 II 类 T6P 合酶 (TPS) 样蛋白有很强的关联。生化和细胞测定表明 TPS 样蛋白作为 SnRK1 的负调节剂发挥作用。除了与 TPS 样蛋白质的稳定相互作用之外，还发现了与已知调节剂的类似错综复杂的联系，这表明植物利用扩展的激酶复合物来微调 SnRK1 活性以获得对代谢应激的最佳反应。