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Post-translational modification of the transcription factor SPATULA by SECRET AGENT and SPINDLY promotes Arabidopsis style development.

ABSTRACT: O-linked-β-N-Acetylglucosamine (O-GlcNAc) and O-fucose are two sugar-based post-translational modifications (PTMs) whose mechanistic role in plant signalling and transcriptional regulation is still largely unknown. Here, we investigated how two O-glycosyltransferase enzymes of Arabidopsis thaliana, SPINDLY (SPY) and SECRET AGENT (SEC) promote the activity of the bHLH transcription factor SPATULA (SPT), during morphogenesis of the plant female reproductive organ apex, the style. SPY and SEC modify N-terminal residues of SPT in vivo and in vitro by attaching O-fucose and O-GlcNAc, respectively. This post-translational regulation does not impact SPT homo- and hetero-dimerisation events, although it enhances the affinity of SPT for the kinase PINOID (PID) gene locus and its transcriptional repression. Our findings offer the first mechanistic example of the effect of O-GlcNAc and O-fucose on the activity of a plant transcription factor and reveal a previously unrecognized roles for SEC and SPY in orchestrating style elongation and shape.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Nicotiana Tabacum (common Tobacco)

TISSUE(S): Leaf

SUBMITTER: Gerhard Saalbach

LAB HEAD: Laila Moubayidin

PROVIDER: PXD043957 | Pride | 2024-01-22

REPOSITORIES: Pride

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Post-translational modification of the transcription factor SPATULA by SECRET AGENT and SPINDLY promotes Arabidopsis style development.

Project description:O-linked-β-N-Acetylglucosamine (O-GlcNAc) and O-fucose are two sugar-based post-translational modifications (PTMs) whose mechanistic role in plant signalling and transcriptional regulation is still largely unknown. Here, we investigated how two O-glycosyltransferase enzymes of Arabidopsis thaliana, SPINDLY (SPY) and SECRET AGENT (SEC) promote the activity of the bHLH transcription factor SPATULA (SPT), during morphogenesis of the plant female reproductive organ apex, the style. SPY and SEC modify N-terminal residues of SPT in vivo and in vitro by attaching O-fucose and O-GlcNAc, respectively. This post-translational regulation does not impact SPT homo- and hetero-dimerisation events, although it enhances the affinity of SPT for the kinase PINOID (PID) gene locus and its transcriptional repression. Our findings offer the first mechanistic example of the effect of O-GlcNAc and O-fucose on the activity of a plant transcription factor and reveal a previously unrecognized roles for SEC and SPY in orchestrating style elongation and shape.

2024-01-22 | PXD044034 | Pride

Post-translational modification of the transcription factor SPATULA by SECRET AGENT and SPINDLY promotes Arabidopsis style development.

2024-01-22 | PXD037917 | Pride

Post-translational modification of the transcription factor SPATULA by SECRET AGENT and SPINDLY promotes Arabidopsis style development.

2024-01-22 | PXD043987 | Pride

E. coli expressed SECRET AGENT O-GlcNAc modifies threonine 829 of GIGANTEA

Project description:The Arabidopsis thaliana glycosyl transferases SPINDLY (SPY) and SECRET AGENT (SEC) modify nuclear and cytosolic proteins with O-linked fucose or O-linked Nacetylglucosamine (O-GlcNAc), respectively. O-fucose and O-GlcNAc modifications can occur at the same sites. SPY interacts physically and genetically with GIGANTEA (GI), suggesting that it could be modified by both enzymes. Previously, we found that, when co-expressed in Escherichia coli, SEC modifies GI; however, the modification site was not determined. By analyzing the overlapping sub-fragments of GI, we identified a region that was modified by SEC in E. coli. Modification was undetectable when threonine 829 (T829) was mutated to alanine, while the T834A and T837A mutations reduced the modification, suggesting that T829 was the primary or the only modification site. Mapping using mass spectrometry detected only the modification of T829. Previous studies have shown that the positions modified by SEC in E. coli are modified in planta, suggesting that T829 is O-GlcNAc modified in planta.

2024-07-15 | PXD053909 | Pride

Quantitative O-GlcNAc analysis between wild type and sec-5 using SILIA (F1 on QE-HF) and Eclipse (HCD)

Project description:O-GlcNAcylation is a crucial post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. Plants have close OGT homologs, SECRET AGENT (SEC) and SPINDLY (SPY). In vitro, SEC has shown O-GlcNAc activity. Recently, a surprising discovery in wheat revealed an atypical TaOGT(TaOGT1) with no structural similarity to SEC and SPY enzyme (Fan et al. 2021). TaOGT1 was found to O-GlcNAcylate TaGRP2. In Arabidopsis, approximately 34 unannotated or uncharacterized proteins share similarity with TaOGT1, leading to questions about whether SEC is the primary contributor to O-GlcNAcylation in plants. Here we use LWAC enrichment and SILIA labeling, quantifying at both MS1. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating SEC’s critical role in mediating O-GlcNAcylation.

2024-05-21 | PXD044668 | Pride

Quantitative O-GlcNAc analysis between wild type and sec-5 using SILIA (F1 on Eclipse (EThcD)

2024-05-22 | PXD044832 | Pride

Quantitative O-GlcNAc analysis between wild type and sec-5 using SILIA (F2 R1 on QE-HF)

2024-05-22 | PXD044831 | Pride

Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

Project description:SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that AtSPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide-repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of AtSPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. AtSPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain dynamically interconverts among inward, middle, and outward states. The entire eleven TPRs are visible in the middle state, with TPR1 of one subunit touching down on the catalytic domain of the other subunit of the AtSPY dimer, whereas the densities of TPRs 1-5 are absent or severely attenuated in the inward and outward states. Analysis of mass spectrometry, co-IP, in planta fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits its catalytic activity, whereas TPRs 1-5 regulate SPY activity by interfering with protein substrate binding.

2023-02-28 | PXD040480 | Pride

SPY mediates O-fucose modification of hundreds of proteins and sugar-dependent growth in Arabidopsis_(Part 2: EtHCD)

Project description:The recent discovery of SPY-catalyzed protein O-fucosylation reveals a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates important roles of SPY in diverse developmental and physiological processes, however, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrate that SPY mediates sugar-dependent growth. We further identify hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in proteomic analyses are undetectable or decreased in the spy mutants and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, hormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked-N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and hormonal responses. Our study identifies numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

2023-05-10 | PXD038491 | Pride

SPY mediates O-fucose modification of hundreds of proteins and sugar-dependent growth in Arabidopsis_(Part 1: HCD)

2023-05-10 | PXD038490 | Pride