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

Nature Plants volume 8, pages 1245–1261 (2022)Cite this article

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Abstract

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.

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Fig. 1: Sucrose repletion to sucrose-starved cells induces a wave of dephosphorylation of SnRK1 substrates.
Fig. 2: SnRK1 protein interactome mapping.
Fig. 3: Network visualization of the integrated SnRK1 interactome.
Fig. 4: Composition, stoichiometry and structure of the core SnRK1 complex.
Fig. 5: Functional characterization of the relationship between TPS II proteins and SnRK1.

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Data availability

The AP–MS and PL MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifiers PXD029833 (AP–MS) and PXD030048 (PL). The protein interactions from this publication have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct97 and assigned the identifier IM-29283. All structure-related data files related to integrative modelling are deposited in the Zenodo repository (10.5281/zenodo.5552311). The data supporting the findings of this study are available at the Figshare digital repository (https://doi.org/10.6084/m9.figshare.20732371)98. Source data are provided with this paper.

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Acknowledgements

This work was funded by a research grant of the Research Foundation—Flanders (FWO) (G011720N) to G.D.J. and F.R. We acknowledge the VIB Proteomics core facility for LC–MS/MS analyses, A. Bleys for help in preparing the manuscript, M. Teige and Be. Würzinger for providing bacterial strains encoding SnAK1 and SnRK1α1 K48M, and T. Beeckman for providing the TPP-A Gateway entry clone.

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  1. Chao Han

    Present address: The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, China

Authors and Affiliations

  1. Ghent University, Department of Plant Biotechnology and Bioinformatics, Ghent, Belgium

    Jelle Van Leene, Dominique Eeckhout, Astrid Gadeyne, Caroline Matthijs, Chao Han, Nancy De Winne, Geert Persiau, Eveline Van De Slijke, Freya Persyn, Toon Mertens, Wouter Smagghe, Daniël Van Damme, Roman Pleskot & Geert De Jaeger

  2. VIB Center for Plant Systems Biology, Ghent, Belgium

    Jelle Van Leene, Dominique Eeckhout, Astrid Gadeyne, Caroline Matthijs, Chao Han, Nancy De Winne, Geert Persiau, Eveline Van De Slijke, Freya Persyn, Toon Mertens, Wouter Smagghe, Daniël Van Damme, Roman Pleskot & Geert De Jaeger

  3. Laboratory for Molecular Plant Biology, Biology Department, KU Leuven, Heverlee-Leuven, Belgium

    Nathalie Crepin, Ellen Broucke & Filip Rolland

  4. KU Leuven Plant Institute—LPI, Heverlee-Leuven, Belgium

    Nathalie Crepin, Ellen Broucke & Filip Rolland

  5. Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic

    Roman Pleskot

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Contributions

J.V.L. wrote the manuscript. J.V.L., D.E., A.G., C.H., D.V.D., F.R. and G.D.J. conceived the research. J.V.L., A.G., C.M., C.H., N.D.W., G.P., E.V.D.S., F.P., T.M., W.S., N.C. and E.B. performed experiments. D.E. and J.V.L. analysed the MS data. R.P. performed the structural analysis.

Corresponding author

Correspondence to Geert De Jaeger.

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Nature Plants thanks Justin Walley, Markus Teige and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sucrose-dependent phosphoproteome analysis.

a, Hierarchical cluster analysis of the sucrose-dependent phosphosites like in Fig. 1c, showing both the sucrose- and TOR-dependent phosphosite intensity fold changes (FC). b, Comparison of phosphopeptide and protein fold changes (Log2FC) for 37 out of the 109 sucrose-dependent phosphorproteins that were identified in a shotgun proteome analysis on the sucrose-starved (t0) and sucrose-replenished (t20) samples. Blue, Log2FC(Proteome), red, Log2FC(Phosphoproteome). For every protein, the corresponding phosphosites are shown between brackets in order of appearance (from left to right). c, (left panel) Bar chart showing the percentage of sites from the sucrose down-, sucrose up-, or TOR-regulated phosphosites in which the central phosphorylated residue is conserved in a given plant species. (right panel) Bar chart showing the fraction of phosphosites for which the ratio of the blosum score of the sequence window over the global blosum score of the whole protein sequence is higher than one.

Extended Data Fig. 2 In vitro kinase assay results.

Full autoradiograms (1 h exposure) and Coomassie stained gel pictures of the kinase assays shown in Fig. 1e. The lower panel shows the absence of phosphorylation with the catalytically inactive SnRK1α1 K48M mutant. In the first lane, the sample from the negative control without substrate was loaded together with the protein ladder. Molecular weights (MW) of the recombinant HisMBP fusions are shown. The experiment was three times independently repeated with similar results.

Extended Data Fig. 3 Dot plot matrix of the 31 bait-specific SnRK1 interactors found in more than one experimental condition.

Dot plot representation is like in Fig. 2b.

Extended Data Fig. 4 Stoichiometry analysis of the remaining SnRK1 subunits.

Results obtained with SnRK1α1, SnRK1β2 or SnRK1β3 as bait proteins are shown like in Fig. 4a.

Extended Data Fig. 5 SnRK1 domain architecture.

a, Structures of the different SnRK1 subunits taken from the Alphafold Structure Database are shown in the ribbon representation. High confident regions are in purple, and low confident regions are in green. α-CTD, C-terminal domain of SnRK1α1; β-CTD, C-terminal domain of SnRK1β2; CBS, cystathionine-synthetase motif; C-term, C-terminus; KD, kinase domain; N-term, N-terminus. b, Three-dimensional multiscale structural representation of the SnRK1 subunits. The individual domains were represented by beads of varying sizes (1 to 20 amino acid residues per bead), arranged into either a rigid body or a flexible string of beads. Numbers in brackets represent a position in the protein sequence.

Extended Data Fig. 6 Convergence, ensemble precision and sampling exhaustiveness of the integrative structure of the heterotrimer composed of SnRK1α1, SnRK1β2 and SnRK1βγ.

a, Convergence of the model score calculated for 9,026 good-scoring models (mean ± SD; n = 10). The scoring did not improve after the addition of more independent models. The red line depicts a lower bound on the total score. b, The sampling precision as defined by three criteria; first, the p-value calculated using the χ2- test for homogeneity of proportions (red dots); second, the effect size for the χ2-test is quantified by the Cramer’s V value (blue squares); third, sufficiently large clusters (containing at least ten models) visualized as green triangles. The vertical dotted gray line indicates the root mean square displacement (RMSD) clustering threshold at which three criteria are satisfied (p-value > 0.05, Cramer’s V < 0.10, and the population of clustered models > 0.80). The sampling precision is thus 30 Å. c, Good-scoring models were split into two populations. Using the sampling precision 30 Å as the threshold, populations of samples 1 (light red) and 2 (blue) form three clusters. 98% of the models belong to cluster 1, which has a precision of 23 Å. d, Localization density maps for sample 1 and sample 2 of cluster 1, visualized here at a threshold equal to one-tenth of the maximum. The cross-correlation of the localization density maps of the two samples is 0.987, indicating that the position of SnRK1 subunits in the two samples is effectively identical at the model precision of 23 Å.

Extended Data Fig. 7 Structural analysis of the core SnRK1 complex composed of SnRK1α2, SnRK1β2 and SnRK1βγ.

a, Convergence of the model score calculated for 15,016 good-scoring models (mean ± SD; n = 10). The scoring did not improve after the addition of more independent models. The red line depicts a lower bound on the total score. b, The sampling precision as defined by three criteria; first, the p-value calculated using the χ2- test for homogeneity of proportions (red dots); second, the effect size for the χ2-test is quantified by the Cramer’s V value (blue squares); third, sufficiently large clusters (containing at least ten models) visualized as green triangles. The vertical dotted gray line indicates the root mean square displacement (RMSD) clustering threshold at which three criteria are satisfied (p-value > 0.05, Cramer’s V < 0.10, and the population of clustered models > 0.80). The sampling precision is thus 25 Å. c, Good-scoring models were split into two populations. Using the sampling precision 25 Å as the threshold, populations of samples 1 (light red) and 2 (blue) form four clusters. 82% of the models belong to cluster 1, which has a precision of 20 Å. d, Localization density maps for sample 1 and sample 2 of cluster 1, visualized here at a threshold equal to one-tenth of the maximum. The cross-correlation of the localization density maps of the two samples is 0.958, indicating that the position of SnRK1 subunits in the two samples is effectively identical at the model precision of 20 Å. e, Structure of the core SnRK1 complex as obtained by the integrative modeling approach. The structure presents a multiscale centroid structure, that is the structure with the minimal sum of root mean square deviations from all the good-scoring models in the dominant cluster 1. f, Input cross-links (gray dashed lines) mapped on the centroid structure. g, SnRK1 domains mapped on the centroid structure. CBM, carbohydrate-binding module; α-CTD, C-terminal domain of SnRK1α2; β-CTD, C-terminal domain of SnRK1β2; CBS, cystathionine-synthetase motif; KD, kinase domain. h, Distance distribution of obtained chemical cross-links in the centroid structure. The dotted red line represents the threshold for the consistent cross-links. i, The residue contact frequency map, calculated over ten best-scoring models, is depicted by colors ranging from white (0, low frequency) to blue (1, high frequency). A contact between a pair of amino acid residues is defined by the distance between bead surfaces below 35 Å. Cross-links are plotted as green dots (consistent cross-links - XLs) or orange dots (inconsistent XLs).

Extended Data Fig. 8 Comparison of the human AMPK and plant SnRK1 structures.

a, Comparison of the arrangements of the AMPK and SnRK1 domains mapped on the centroid structures. b, Multiple sequence alignment of the αG-helix from yeast, mammalian and Arabidopsis SNF1/AMPK/SnRK1 catalytic subunits. The cross-linked tryptic peptide that connects both SnRK1 α-subunits and the cross-linked lysine residues are indicated. The proximal hydrophobic residues that are involved in the dimerization of the catalytic subunits are marked by a blue rectangular. Protein sequences were extracted from the UNIPROT database and aligned in Jalview.

Supplementary information

Supplementary Information

Supplementary Note and references.

Reporting Summary

Supplementary Table

Supplementary Tables 1–8.

Source data

Source Data Fig. 1

Unprocessed immunoblots related to Fig. 1f.

Source Data Fig. 5

Stain-free loading controls and unprocessed immunoblots related to Fig. 5b–d.

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Van Leene, J., Eeckhout, D., Gadeyne, A. et al. Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins. Nat. Plants 8, 1245–1261 (2022). https://doi.org/10.1038/s41477-022-01269-w

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