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bioRxiv · 10.64898/2026.01.16.700008

Proteomic Analysis Reveals Widespread Regulation of Substrate Protein Abundance by O-fucosylation and O-GlcNAcylation

Karunadasa, S. S.·Reyes, A. V.·Grismer, T. S.·Shrestha, R.·Byun, D.·Carey, S.·Ni, W.·Xu, S.-L.
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Abstract

O-glycosylation of nucleocytosolic proteins by the Arabidopsis enzymes SPINDLY (SPY; O-fucosyltransferase) and SECRET AGENT (SEC; O-GlcNAc transferase) is essential for plant growth and development, yet the scope of their substrates and regulatory impact remains poorly defined. Here, we combined TurboID-based proximity labeling with quantitative proteomics to systematically map the SPY interactome and determine how SPY- and SEC-dependent modifications influence protein abundance. A functional SPY-TD enriched 221 proxiome proteins, including 80 known O-fucosylated substrates and 141 new interactors. The SPY-TD proxiome is enriched in nuclear pore components, chromatin regulators, transcription factors, and RNA-processing proteins. Integration with O-fucose and O-GlcNAc datasets yielded a comprehensive Arabidopsis SPY/SEC (At-S/S) protein list of 886 candidates. We quantified proteome-wide changes in spy single mutants and inducible spy sec double mutants. Loss of SPY alone caused selective stabilization or destabilization of targets, whereas combined SPY/SEC depletion triggered widespread, synergistic protein abundance changes, particularly affecting nucleoporins, transcriptional regulators, and RNA-binding proteins. Integration with ubiquitination datasets revealed extensive overlap, supporting potential crosstalk between O-fucosylation, O-GlcNAcylation, and ubiquitin-mediated protein turnover. Together, our study establishes proximity labeling as a powerful strategy to define plant O-glycosylation networks and reveals dual, context-dependent roles of SPY and SEC in controlling protein homeostasis and stress-responsive pathways.

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Karunadasa, S. S., Reyes, A. V., Grismer, T. S., Shrestha, R., Byun, D., Carey, S., Ni, W., Xu, S.-L.. 2026-01-19. Proteomic Analysis Reveals Widespread Regulation of Substrate Protein Abundance by O-fucosylation and O-GlcNAcylation. https://doi.org/10.64898/2026.01.16.700008

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