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A MULTI-DIMENSIONAL INTERACTOMICS VIEW ON THE PLANT SNRK1 KINASE COMPLEX REVEALS A KEY REGULATORY ROLE FOR THE CLASS II T6P SYNTHASE-LIKE PROTEINS – part2 TurboID-MS dataset

ABSTRACT: The plant SNF1-related kinase (SnRK1) plays a central role in energy and metabolic homeostasis. SnRK1 controls growth upon activation by energy-depleting stress conditions, while also monitoring key developmental transitions and nutrient allocation between source and sink tissues. To obtain deeper insight into the SnRK1 complex and its upstream regulatory mechanisms, we explored its protein interaction landscape in a multi-dimensional setting, combining affinity purification, proximity labeling and crosslinking mass spectrometry. Integration of these analyses not only offers a unique view on the composition, stoichiometry and structure of the core heterotrimeric SnRK1 complex but also reveals a myriad of novel robust SnRK1 interactors.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Cell Culture

SUBMITTER: Dominique Eeckhout

LAB HEAD: Geert De Jaeger

PROVIDER: PXD030048 | Pride | 2022-08-30

REPOSITORIES: Pride

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A MULTI-DIMENSIONAL INTERACTOMICS VIEW ON THE PLANT SNRK1 KINASE COMPLEX REVEALS A KEY REGULATORY ROLE FOR THE CLASS II T6P SYNTHASE-LIKE PROTEINS – part1 AP-MS dataset

Project description:The plant SNF1-related kinase (SnRK1) plays a central role in energy and metabolic homeostasis. SnRK1 controls growth upon activation by energy-depleting stress conditions, while also monitoring key developmental transitions and nutrient allocation between source and sink tissues. To obtain deeper insight into the SnRK1 complex and its upstream regulatory mechanisms, we explored its protein interaction landscape in a multi-dimensional setting, combining affinity purification, proximity labeling and crosslinking mass spectrometry. Integration of these analyses not only offers a unique view on the composition, stoichiometry and structure of the core heterotrimeric SnRK1 complex but also reveals a myriad of novel robust SnRK1 interactors.

2022-08-30 | PXD029833 | Pride

NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis

Project description:Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like-1 (AtEPL1) and Esa1- Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

2021-12-20 | PXD030302 | Pride

Mapping the protein interaction network of adaptor protein complex in Arabidopsis identifies P34 as a common stability regulator

Project description:Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargos and coated vesicle accessory proteins. Since in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargos of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins, and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

2023-12-07 | PXD035454 | Pride

Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing

Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. Identification of RNA polymerase active sites in Drosophila S2 cell line using PRO-seq method. Identification of transcription initiation sites in Drosophila S2 cell line using PRO-cap method. Identification of changes in RNA polymerase active sites on transgenic Hsp70 promoters upon disruption of DNA sequence elements in 3 transgenic fly lines using PRO-seq method.

2013-02-01 | E-GEOD-42117 | biostudies-arrayexpress

Global Interferon-stimulated Genes Interactome Network

Project description:Interferon-stimulated genes (ISGs) form the backbone of innate immune system and are pivotal for limiting intra- and intercellular viral replication and spread. We conducted a mass spectrometry–based survey to understand the fundamental organization of the innate immune system and to explore molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify novel regulators of immunity and immune system–related processes. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems in order to efficiently fight viral infections.

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FluAV vRNP interactome - A protein-interaction network of interferon-stimulated genes extends the innate immune system landscape

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Characterization of the starvation response in the Arabidopsis dcl1-9 mutant

Project description:The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to the well-established role of miRNAs as regulators of plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1 activation. To test this we have performed comparative analyses of the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis. To assess the impact of miRNA deficiency on the starvation response we performed transcriptomics analyses of WT and dcl1-9 plants by subjecting leaves to 6h of light (control) or darkness (starvation)

2013-07-15 | E-GEOD-46713 | biostudies-arrayexpress

A conserved signaling complex controls warm temperature-mediated plant growth

Project description:The model plant Arabidopsis thaliana responds to mild high temperature by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. Our understanding of the genetic regulation of thermomorphogenesis has increased in recent years. However, hardly anything is known about molecular mechanisms outside A. thaliana and cellular signaling pathways have been underexplored. Therefore, we mapped changes in protein phosphorylation in A. thaliana and crops exposed to warm temperature. Based on these results, we identified and characterized a novel, functionally conserved signaling complex of MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASEs (MAP4KS) in warm temperature-mediated growth regulation in plants. This contributes to our understanding of warm temperature signaling, and can help guarantee food security under a changing climate

2021-03-25 | PXD015483 | Pride

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

Project description:Clathrin-mediated endocytosis is an essential cellular internalisation pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET/TPLATE complex (TPC) plays an essential, but not well-defined role in endocytosis in plants. Here, we show that two highly disordered TPC subunits, AtEH1 and AtEH2 function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, early-, and late-stage endocytic accessory proteins. Importantly, clathrin forms ordered assemblies within the condensate environment. Biomolecular condensation therefore acts to promote dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, the disordered region sequence properties of AtEH1 regulate the material properties of the endocytic condensates in vivo. Alteration of the material properties influences endocytosis dynamics, and thereby impairs environmental adaption. In summary, our findings reveal how collective interactions shape endocytosis.

2023-12-07 | PXD045068 | Pride

Quantitative proteomics reveals SnRK1-dependent protein in rice sheaths during grain filling

Project description:Insufficient leaf photosynthesis promotes sheath NSC transport to the panicle during grain filling. SnRK1 regulates assimilate distribution between plant tissues and organs. However, the mechanism by which SnRK1 regulates sheath NSC transport to the panicle and its response to limited leaf assimilation remain unclear. Here, we performed leaf cutting (LC) at anthesis to simulate insufficient leaf assimilation and set no treatment as a CK. In response to LC, SnRK1 activity increased rapidly, and NSC transport was advanced. Sheath NSCs were not transported in a snrk1a mutant with deficient SnRK1 activity. These results indicated that SnRK1 activity is important for sheath NSC transport. The high correlation of T6P and sucrose and the inhibition of SnRK1 by T6P in sheaths in vitro implied that T6P slightly inhibits SnRK1 activity in rice sheaths in response to sucrose availability. The increase in SnRK1 activity was accompanied by an increase in OsSnRK1a expression and a low sucrose content. Low sucrose signaling increases SnRK1 transcription. Therefore, we speculated that OsSnRK1a expression positively regulates SnRK1 activity in response to low sucrose availability in rice sheaths. Through phosphoproteomics and further PRM verification, 20 phosphosites that depend on SnRK1 and are correlated with NSC transport were obtained. Based on the function of these sites and proteins, we found that SnRK1 regulates starch degradation, sucrose metabolism, phloem transport, sugar transport across tonoplast, and glycolysis via phosphorylation to promote sheath NSC transport. Overall, our results revealed the importance, function and regulatory mechanism of SnRK1 in sheath NSC transport.

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