Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins.

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.

Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase.

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.