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O-Fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling during Seed Germination and Seedling Development--Partial Submission

ABSTRACT: We utilized ETD (Electron Transfer Dissociation)-MS/MS analysis to identify O-fucosylation sites in ectopically expressed CPN20 in mammalian cells and Arabidopsis.

INSTRUMENT(S): LTQ Orbitrap Elite

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

SUBMITTER: Wen Zhou

LAB HEAD: Wen Zhou

PROVIDER: PXD027272 | Pride | 2022-02-17

REPOSITORIES: Pride

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			Action	DRS
	17-030_LL0209_1.dat	Other
	17-030_LL0209_1.mgf	Mgf
	17-030_LL0209_1.mzid	Mzid
	17-030_LL0209_1_.raw	Raw
	17-221_LL0704_3.dat	Other

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Publications

O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development.

Liang Lin L Wang Qi Q Song Zihao Z Wu Yaxin Y Liang Qing Q Wang Qingsong Q Yang Jinli J Bi Ying Y Zhou Wen W Fan Liu-Min LM

Frontiers in plant science 20211012

SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation site ...[more]

PMID: 34712252

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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

Phosphorylation Promotes DELLA Activity by Enhancing Its Binding to Histone H2A at Target Chromatin in Arabidopsis

Project description:DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via its GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)-conjugation to alter its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O-fucosylation, but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear. Here, we identified phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by tandem mass spectrometry analysis, and showed that phosphorylation of the RGA LKS-peptide in the poly-S/T region enhances RGA-H2A interaction and RGA association with target promoters. Interestingly, phosphorylation does not affect RGA-TF interactions. Our study has uncovered that phosphorylation is a new regulatory mechanism of DELLA activity.

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Optimized mucin-selective enrichment strategy to probe the mucinome

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O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development.

Project description:SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.

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