Biology subjects

Karunadasa, S. S.

Publications and source records attributed to Karunadasa, S. S..

3 recordsLinked to original sources

bioRxiv2026

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

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.

systems biology↗

bioRxiv2025

Mapping Architecture of Protein complexes in Arabidopsis using XL-MS

Capturing molecular machines in action is essential for understanding protein complex architecture, cellular regulation, and gene function. Here, we present a large-scale structural proteomics resource for Arabidopsis thaliana generated using an optimized cross-linking mass spectrometry (XL-MS) workflow. Using the trifunctional cross-linker PhoX, whose phosphonic acid moiety enables immobilized metal affinity chromatography (IMAC)-based enrichment, we selectively enriched cross-linked peptides from whole-cell lysates, chloroplasts, and nuclei. Analysis with pLink 3.2 identified 52,944 unique cross-linked peptide pairs, corresponding to 37,531 residue-level contacts across 5,064 proteins. These data define 3,083 protein-protein interactions, including 2,385 heteromeric and 698 homomultimeric interactions. Comparison with the STRING database showed that 676 interactions are supported by STRING scores [≥]0.9. Structural mapping to Protein Data Bank (PDB) and AlphaFold models showed that most cross-links were within the expected 35 [A] distance constraint. The dataset further enabled the analysis of protein connectivity and complex topology across diverse molecular assemblies, including the Rubisco holoenzyme, chloroplast 70S ribosome, photosystem complexes, and the cytosolic 80S ribosome together with associated biogenesis and regulatory factors. We also identified histone-associated complexes, including interactions involving an O-acyltransferase. By providing residue-level structural constraints for a substantial portion of the Arabidopsis proteome, this study provides a resource for exploring plant molecular machines and their spatial organization. Significance StatementUnderstanding how proteins interact within living cells is essential to deciphering cellular architecture and function. However, capturing native protein-protein interactions (PPIs) on a global scale has proven technically challenging. Here, we present a proteome-wide cross-linking mass spectrometry (XL-MS) platform that can systematically map direct PPIs in plant cells without requiring transgenic manipulation. This approach identifies thousands of interactions spanning major subcellular compartments and characterizes the in situ organization of critical protein assemblies, such as photosystems, ribosomes, and chromatin-associated connectivity. By mapping both established and less-characterized interactions, this work advances our understanding of the plant protein interactome and provides a valuable resource for investigating the structural organization of the plant proteome.

molecular biology↗

bioRxiv2025

Next-Generation Mapping of the ACINUS-Mediated Alternative Splicing Machinery and Its Regulation by O-glycosylation in Arabidopsis

Alternative splicing (AS) is a key mechanism of gene regulation, but the full repertoire of proteins involved and the regulatory mechanisms governing this process remain poorly understood. Using TurboID-based proximity labeling coupled with mass spectrometry (PL-MS), we comprehensively mapped the Arabidopsis AS machinery, focusing on the evolutionarily conserved splicing factor ACINUS, its paralog PININ, and the stable interactor SR45. We identified 298 high-confidence components, including both established and novel interactors, providing strong evidence that alternative splicing is coupled to transcription and that multiple RNA processing steps occur simultaneously in plants. Bioinformatic analysis reveals high redundancy, conserved mechanisms, and unique plant-specific features. Selected known and novel interactors were validated by AS readouts and phenotypic analysis, which also revealed a coordinated influence on splicing. Furthermore, a systematic evaluation of O-glycosylation double mutants revealed that SECRET AGENT (O-GlcNAc transferase) and SPINDLY (O-fucose transferase) modulate AS through both ACINUS-dependent and -independent pathways. Our results reveal the conserved as well as plant-specific AS regulatory network and highlight the global role of sugar modification in RNA processing.

plant biology↗