SPINDLY mediates O-fucosylation of hundreds of proteins and sugar-dependent growth in Arabidopsis

Abstract

The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed 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 demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically 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, phytohormone 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 phytohormone responses. Our study identified 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.

SECRET AGENT O-GlcNAc Modifies GIGANTEA: Methods for Mapping of O-Linked β-N-Acetylglucosamine Modification Sites Using Mass Spectrometry

“…Consistent with SEC and SPY having overlapping functions, O-GlcNAc and O-fucose modifications can occur at the same position (Bi et al, 2023;Xu et al, 2017).…”

“…sec spy double mutants are embryo lethal suggesting that SEC and SPY have overlapping functions (Hartweck et al, 2002). Recent surveys have identified numerous O-GlcNAc and O-fucose modified plant protein (Bi et al, 2023;Li et al, 2023;Wu et al, 2022;Xu et al, 2017;Zentella et al, 2023).…”

Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

“…13), lending further support to the proposed mechanism of a substrate-assisted SN2 reaction proposed here and by Zhu et al 39 . As recent proteomic analysis of SPY substrates did not reveal a distinct pattern around the O-fucosylation sites, except the enrichment of serine residues 37,38 , how SPY selectively recognizes diverse peptide sequences requires further investigation. Intriguingly, although our truncation study also suggested a model of self-inhibition from the N-terminal disordered peptide (residues 1-39) in SPY, we did not observe any evidence of the T-shaped trans inhibitory state within a dimer of SPY dimer as observed in the crystal structure.…”

“…4d), suggesting that the rigid conformation with 11 TPRs locking the catalytic domain may not be optimal for the SPY-RGA interaction and catalysis. Furthermore, as the donor substrate GDP-fucose is largely recognized by the catalytic domain alone, whereas the recipient substrate peptide is tethered by the Asn ladder within the TPRs, movement of the catalytic domain with the bound GDP-fucose may dynamically position GDP-fucose close to the receptor serine/threonine residues from diverse substrate sequences 37,38 in the active site for efficient catalysis despite the local variation of the substrate binding mode caused by the different peptide sequences.…”

Sugar and SPY(ce): Large-scale identification of SPINDLY-dependentO-fucosylation targets in Arabidopsis

“…In this issue of The Plant Cell , Yang Bi, Ruben Shrestha and colleagues ( Bi et al 2023 ) identified a large number of O -fucosylated proteins from Arabidopsis by affinity purification. The authors first showed that spy mutants grew poorly on sucrose-supplemented media in the dark (see Figure ), indicating a crucial role for SPY in sugar-dependent growth.…”