SPINDLY o-fucosylates nuclear and cytoplasmic proteins involved in diverse cellular processes in plants

Authors: Zentella, Rodolfo; WANG, YAN - Duke University; ZAHN, EMILY - University Of Virginia; HU, JIANHONG - Duke University; JIANG, LIANG - Duke University; SHABANOWITZ, JEFFREY - University Of Virginia; HUNT, DONALD - University Of Virginia; SUN, TAI-PING - Duke University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2022
Publication Date: 2/6/2023
Citation: Zentella Gomez, R., Wang, Y., Zahn, E., Hu, J., Jiang, L., Shabanowitz, J., Hunt, D., Sun, T. 2023. SPINDLY o-fucosylates nuclear and cytoplasmic proteins involved in diverse cellular processes in plants. Plant Physiology. 191(3):1546-1560. https://doi.org/10.1093/plphys/kiad011.
DOI: https://doi.org/10.1093/plphys/kiad011

Interpretive Summary: SPINDLY (SPY) is a novel enzyme that modifies target proteins by attaching the sugar fucose to specific amino acids, namely Serine and Threonine. Previous genetic studies in the model plant Arabidopsis thaliana indicate that targets of AtSPY are involved in various aspects of plant development, modulating plant hormone responses, circadian rhythm, and plant responses to biotic and abiotic stresses. However, very few SPY targets have been identified. This work reports the identification of 88 SPY targes from Arabidopsis and tobacco by purification of modified target proteins with a protein that specifically recognizes the fucose modification. The identity of the purified proteins was determined by tandem mass spectrometry. The majority of the AtSPY targets are nuclear proteins that function in DNA repair, gene expression and transport between nucleus and cytoplasm. Other AtSPY targets are involved cell growth and division. An enzyme related to AtSPY, known as OGT, adds a different sugar, N-acetylgucosamine (GlcNAc), also to Serines and Threonines of its targets. A comparison of the collection of reported GlcNAc-modified proteins with the Fucose-modified proteins found in this work, revealed that 30% of AtSPY targets are also GlcNAc-modified, indicating that these distinct sugar modifications can co-regulate many protein functions. This study provides a valuable resource to study the regulatory mechanisms involved with these modifications.

Technical Abstract: SPINDLY (SPY) is a novel nucleocytoplasmic protein O-fucosyltransferase that regulates target protein activity or stability via O-fucosylation of specific Ser/Thr residues. Previous genetic studies indicate that AtSPY regulates plant development during vegetative and reproductive growth by modulating gibberellin and cytokinin responses. AtSPY also regulates the circadian clock and plant responses to biotic and abiotic stresses. The pleiotropic phenotypes of spy mutants point to the likely role of AtSPY in regulating key proteins functioning in diverse cellular pathways. However, very few AtSPY targets are known. Here, we identified 88 SPY targets from Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana via the purification of O-fucosylated peptides using Aleuria aurantia lectin followed by electron transfer dissociation-MS/MS analysis. Most AtSPY targets were nuclear proteins that function in DNA repair, transcription, RNA splicing, and nucleocytoplasmic transport. Cytoplasmic AtSPY targets were involved in microtubule-mediated cell division/growth and protein folding. A comparison with the published O-linked-N-acetylglucosamine (O-GlcNAc) proteome revealed that 30% of AtSPY targets were also O-GlcNAcylated, indicating that these distinct glycosylations could co-regulate many protein functions. This study unveiled the roles of O-fucosylation in modulating many key nuclear and cytoplasmic proteins and provided a valuable resource for elucidating the regulatory mechanisms involved.