Biology subjects

Grismer, T. S.

Publications and source records attributed to Grismer, T. S..

4 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

Cellulose Synthase Complex and Remorin Nanodomains Mediate Stress Resilience Through Cell Wall-Plasma Membrane Attachments

HighlightsO_LIThe plasma membrane forms attachments to the plant cell wall that are revealed by hyperosmotic shock and correlate with tolerance to stress. C_LIO_LICellulose Synthase Complex (CSC) clusters and REMORIN (REM) nanodomains localize to cell wall-plasma membrane attachment sites. C_LIO_LICSC density at the plasma membrane determines the extent of cell wall-plasma membrane attachment under hyperosmotic stress. C_LIO_LIREMs rapidly form nanodomains under hyperosmotic stress and are associated with SHOU4/4L, CSC exocytosis inhibitors that limit CSC density at the plasma membrane. C_LI The outer cell surface of an organism is the frontline for detecting and responding to environmental stimuli. In plants, this interface consists of the plasma membrane that lies beneath the cell wall and remains associated with it through attachment sites. These wall-membrane attachments become evident upon hyperosmotic shock, when severe water loss causes the membrane to retract from the wall. Despite their long-standing observation, the molecular identity and function of these attachments remain poorly understood. Here, we identified two mechanisms governing wall-membrane attachments: one dependent on the Cellulose Synthase Complex (CSC), whose density at the plasma membrane positively correlates with resistance to hyperosmotic stress, and the other on REMORIN (REM), which acts antagonistically to the CSC mechanism. Using proximity-labeling proteomics, we identified SHOU4/4L as REM-associated proteins that mediate this antagonism. Together, our findings reveal how wall-membrane attachments are patterned to mediate plant cell resilience under water stress.

plant biology↗

bioRxiv2024

Impact of alternative splicing on Arabidopsis proteome

Limited proteomic evidence makes it unclear to what extent alternative splicing (AS) isoforms are translated and functionally relevant in eukaryotes. Here, we present a comprehensive proteomic analysis in plants using large-scale data mining, extensive fractionation of AspN-and trypsin-digested proteomes, and both label-free and TMT labeling. In total, we identified 471,196 peptides from 22,479 proteins by searching against Araport11, revealing 32,110 isoform-specific peptides. Using an integrated proteogenomic workflow coupled with SUPPA, we classified these peptides into 2,442 AS events, 879 of which involved intron retention (IR). Further analysis of unannotated events revealed 91 additional IRs that are translated, confirming that retained introns can give rise to peptides. AlphaFold modeling predicted the structural and functional impacts of these isoforms. Our dataset improved existing gene model annotations. By comparing wild-type plants with the AS mutant acinus pinin, we found that IR regulates transcript and protein abundance nonlinearly. Phenotypic assays confirmed the functional consequences, including reduced chlorophyll, impaired growth, and increased anthocyanin. Take together, our results provide large-scale proteomic evidence that AS isoforms are translated in plants, demonstrating how AS diversifies the proteome, regulates protein abundance, and affects growth and development.

plant biology↗

bioRxiv2024

Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration [≤] 50 {micro}M is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipes MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

molecular biology↗