bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.04.28.651104

Mapping Architecture of Protein complexes in Arabidopsis using XL-MS

Trinh, C. S.·Shrestha, R.·Conner, W. C.·Reyes, A. V.·Karunadasa, S. S.·Liu, G.·Hu, K.·Xu, S.-L.
View original source ↗Export metadata ↓

Abstract

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.

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Trinh, C. S., Shrestha, R., Conner, W. C., Reyes, A. V., Karunadasa, S. S., Liu, G., Hu, K., Xu, S.-L.. 2025-04-28. Mapping Architecture of Protein complexes in Arabidopsis using XL-MS. https://doi.org/10.1101/2025.04.28.651104

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

An ssRNA Phage Single-gene Lysis Protein Targeting MraY Reveals Convergent Evolution Across the RNA-DNA Phage Boundary

Small lytic phages of the ssRNA Leviviricetes and ssDNA Microviricetes achieve host lysis by expression of a single protein, designated as an Sgl protein. Four ssRNA Sgls have been shown to inhibit steps in peptidoglycan biosynthesis: three target MurJ, the Lipid II flippase, and one targets MurA, the committed step of the pathway. A fifth Sgl, the E protein of the canonical ssDNA microvirus {varphi}X174, inhibits MraY, which catalyzes the synthesis of the first lipid-linked intermediate. This inhibition requires SlyD, a cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase. The Sgl of the Caulobacter ssRNA phage {varphi}Cb5 (Cebevirus halophobicum) has not been characterized. Here, we present physiological evidence that Sgl{varphi}Cb5 targets MraY. Moreover, we show that, despite the absence of detectable sequence similarity, Sgl{varphi}Cb5 shares several features with the {varphi}X174 E protein, including the distribution of hydrophobic and charged residues, a proposed N-out, C-in membrane topology, and retention of lytic activity in an Sgl{varphi}Cb5 -LacZ fusion. Furthermore, expression of the Caulobacter cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase CC2799 enhances Sgl{varphi}Cb5 -mediated lysis in E. coli. These parallels between Sgl function in a DNA phage and an RNA phage either represent remarkably consonant convergent evolution or some evolutionary cross-over event for which there is currently no precedent.

molecular biology↗
bioRxiv2026

HERC1-mediated orphan subunit quality control maintains proteasome homeostasis and is disrupted in HERC1-associated neurodevelopmental disease

Biallelic hypomorphic variants in the E3 ubiquitin ligase HERC1 cause a neurological disorder clinically characterized by macrocephaly, dysmorphic facies, and psychomotor delay. Identifying the substrates of HERC1 is essential for understanding its biological functions and the mechanisms underlying disease-associated variants. To address this, we performed a quantitative proteomic analysis using biotinylated ubiquitin to identify HERC1 targets and examine their regulation in cells expressing HERC1 with or without ubiquitin ligase activity. This approach revealed a broad set of HERC1-dependent ubiquitylation substrates, including proteins from major multiprotein complexes such as the proteasome, the translation initiation machinery, and pathways involved in cell metabolism. Focusing on the proteasome, the most prominently represented complex, we identified several regulatory particle subunits, including PSMC6, PSME1, and PSME2. Specifically, within the 19S regulatory particle, we demonstrated that ubiquitylation of the PSMC6 subunit involves the formation of K48-linked polyubiquitin chains and depends on HERC1 activity. Other 19S subunits, such as PSMC2, PSMC4, and PSMC5, undergo a similar modification. Mechanistically, we show that HERC1 selectively recognizes unassembled subunits, promotes their ubiquitylation, and targets them for proteasomal degradation. Notably, in HERC1-deficient cells, disruption of this mechanism impaired proteasomal activity. At the disease level, quantitative proteomic analysis of the pathogenic HERC1 variant c.1448G>A (p.Gly483Glu) likewise identified altered ubiquitylation of proteasome subunits and components of the translation initiation machinery. Biochemical analyses further revealed impaired ubiquitylation of PSMC6 by the mutant HERC1 protein, associated with a loss of interaction between HERC1 and PSMC6. Together, these findings establish a link between HERC1-associated neurological disorders and defective ubiquitylation pathways. They elucidate the molecular mechanisms by which HERC1 regulates proteostasis and demonstrate how disruption of these processes contributes to disease pathogenesis, thereby highlighting potential avenues for therapeutic intervention.

molecular biology↗
bioRxiv2026

PKR inhibition prevents maladaptive responses to endoplasmic reticulum (ER) stress in early-onset dystonia DYT-PRKRA.

DYT-PRKRA is a rare, childhood-onset dystonia caused by mutations in the PRKRA gene, which encodes the protein PACT. PACT activates protein kinase R (PKR) in response to endoplasmic reticulum (ER) stress. In DYT-PRKRA patient cells, the PACT-PKR interaction is enhanced, increasing sensitivity to ER stress-induced apoptosis. The canonical protein kinase activated by ER stress is PKR-like endoplasmic reticulum kinase (PERK), which phosphorylates the subunit of eukaryotic translation initiation factor 2 (eIF2) to initiate the integrated stress response (ISR) and restore cellular homeostasis via the transcription factor ATF4. However, under persistent stress, ATF4 induces CHOP, shifting the adaptive response toward apoptosis. PKR, also activated by ER stress, phosphorylates eIF2, but its contribution to cellular homeostasis remains understudied. This work shows, for the first time, that PKR activation is essential for CHOP induction during ISR, directly linking enhanced apoptosis in DYT-PRKRA cells to prolonged PKR activation and heightened CHOP induction. Strikingly, a small-molecule PKR inhibitor effectively blocks CHOP expression and significantly decreases apoptosis in DYT-PRKRA cells, suggesting PKR inhibition as an effective therapeutic strategy for DYT-PRKRA.

molecular biology↗