Biology subjects

van Wijk, K. J.

Publications and source records attributed to van Wijk, K. J..

10 recordsLinked to original sources

bioRxiv2026

The chloroplast CLP chaperone-protease system controls the steady-state abundance of the singlet oxygen sensors EXCUTER 1 and 2

O_LIThe chloroplast CLP chaperone-protease is essential for chloroplast biogenesis. CLP substrate selection is aided by the N-recognin CLPS1 and CLPF adaptors. They interact with each other and the CLPC1 chaperone, but their specific functions are poorly understood. C_LIO_LIWe employed in vivo CLPC1 substrate-trapping in Arabidopsis by expressing a 35S:CLPC1-TRAP-STREPII transgene in wild-type (WT), clpf, clps1, and clpfclps1 to test the consequences of the loss of these adaptors on CLPC1-trapped proteins. Immunoblotting and protein half-life experiments were carried out for identified CLP substrates. C_LIO_LIExpression of the 35S:CLPC1-TRAP-STREPII in clps1cpf was embryo lethal. CLPF was trapped at a reduced level in clps1, supporting CLPS-CLPF interactions. Chloroplast 1O2 sensor EXECUTER1 (EX1) was trapped in WT and clps1 but not significantly in clpf. Steady-state protein accumulation of EX1 and its homolog EX2 increased 30-fold in the CLPC1-TRAP lines and clpr2-1, but not in clpf or clps1. In planta experiments showed that the half-life of EX1 is [~]3-fold longer in clpc1-1 than in WT, but EX1 half-life was unaffected in clpf. C_LIO_LIWe conclude that the CLP system plays a key role in EX1,2 homeostasis by keeping their intra-chloroplast concentrations low through continuous degradation, upstream of their 1O2 signaling function. C_LI

plant biology↗

bioRxiv2026

Loss of Mitochondrial FMT Restores Chloroplast Proteostasis via Inter-organelle Compensation

Chloroplast proteostasis is vital for plant development, yet whether cells can actively reprogram organelle communication to restore plastid function when essential protease components fail remains unclear. Using a forward genetic suppressor screen in Arabidopsis, we identify loss of the mitochondria-associated protein FRIENDLY MITOCHONDRIA (FMT) as a strong suppressor of the virescent and growth-retarded clpc1 mutant, which lacks the major chloroplast Clp chaperone ClpC1. Suppression is highly specific and occurs independently of GUN1-mediated retrograde signaling. Integrated multi-omics analyses reveal that clpc1 and fmtclpc1 represent two distinct organelle signaling states. In clpc1, loss of ClpC1 triggers a plastid stress state characterized by repression of photosynthesis-associated transcription factors, induction of plastid metabolic stress markers, and impaired proteolytic activity. By contrast, loss of FMT shifts the system into a recovery state despite persistent mitochondrial clustering. Mechanistically, FMT negatively regulates CLPC2, a ClpC1 paralog, and fmt-mediated rescue results from CLPC2 derepression. Moderately elevated ClpC2 restores in vivo proteolysis, as evidenced by recovery of PAA2 substrate turnover, normalization of chloroplast ultrastructure, and reactivation of photosynthesis-related gene expression. Transcriptomic and proteomic profiling further reveal coordinated remodeling of nuclear gene expression and chloroplast protein investment in the recovery state, including reduced cytosolic folding stress and selective induction of jasmonic acid- and salicylic acid-associated signaling networks. Genetic analyses establish that REC1 and REC2 are required for full CLPC2 induction and phenotypic recovery. Together, our findings uncover a latent inter-organelle compensatory mechanism in which mitochondrial perturbation reprograms nuclear gene expression to restore chloroplast proteostasis when ClpC1 function is compromised.

plant biology↗

bioRxiv2026

Structural basis for dimerization, catalytic regulation, and substrate selectivity in S9D proteases

S9 proteases are widely distributed across the tree-of-life and play essential roles in protein processing. However, the structural and mechanistic basis for protease activity in the S9D subfamily has remained unknown. Here, we report the first high-resolution cryo-EM structures of an S9D protease, chloroplast glutamyl endopeptidase (CGEP) from Arabidopsis thaliana, expressed in plants and bacteria. CGEP adopts a dimeric architecture stabilized by two distinct interfaces: hydrophobic interactions between catalytic domains and an interdomain {beta}-sheet linking the cap and catalytic domains. These interactions create a rigid scaffold that supports a hinge loop, which acts as a steric gate to restrict substrate access and confine catalytic activity to the closed conformation. Unlike S9A-B-C proteases, CGEP maintains an intact catalytic triad in both open and closed states, relying on hinge-loop gating rather than catalytic disruption for regulation. Structural analysis and mutagenesis reveal that the hinge loop forms a conserved pocket favoring glutamate side chains, explaining CGEPs strong glutamate preference at cleavage sites. Together, these findings uncover a unique regulatory paradigm for S9D proteases and provide a structural framework for understanding substrate selectivity and dimerization. Significance statementThis study provides the first structural and mechanistic insights into plant S9D proteases, revealing a unique regulatory paradigm that combines hinge-loop gating with substrate-specific recognition. By uncovering how CGEP maintains catalytic integrity while restricting activity to the closed conformation and explaining its strong glutamate preference, these findings advance our understanding of protease diversity and open new avenues for engineering proteases with tailored specificity for agricultural and biotechnological applications.

biochemistry↗

bioRxiv2024

Stromal DUF760-1 and DUF760-2 proteins are degraded by the chloroplast Clp protease system but have very different half-lives

The chloroplast AAA+ chaperone CLPC1 aids to select, unfold and deliver hundreds of proteins to the stromal CLP protease core complex for degradation. Through in vivo CLPC1 trapping we previously identified dozens of trapped proteins that are substrate adaptors (e.g. CLPS1 and CLPF), other chaperones (CLPC2 and CLPD) or (potential) substrates (e.g. RH3) for the CLP chaperone-protease system. Here we show that two of these highly trapped proteins, DUF760-1 and DUF760-2, are substrates for the chloroplast CLP protease system. In planta BiFC and yeast-2-hybrid analyses show that both DUF760 proteins can directly interact with the N-domain of CLPC1. Immunoblotting and confocal microscopy analysis demonstrates that both DUF760 proteins are highly enriched in clpc1-1 and clpr2-1 loss-of function mutants. In vivo cycloheximide chase assays in different genetic backgrounds show that DUF760-1 and 2 are both degraded by the chloroplast CLP protease. The half-life of DUF760-1 is 4-6 hours, whereas DUF760-2 is so unstable that it is very hard to detect unless degradation is inhibited in CLP loss-of-function alleles. Null mutants for DUF760-1 and DUF760-2 show weak but differential pigment phenotypes and differential sensitivity of protein translation inhibitors. The functions of these DUF760 proteins are unknown; the lack of shared mRNA co-expressors and the large difference in half-life and protein abundance, suggest that they play different roles within the chloroplast. Taken together, our results demonstrate that DUF760-1 and 2 are newly discovered substrates of CLP chaperone-protease system.

plant biology↗

bioRxiv2023

The Zea mays PeptideAtlas; a new maize community resource

We developed the Maize PeptideAtlas resource (www.peptideatlas.org/builds/maize) to help solve questions about the maize proteome. Publicly available raw tandem mass spectrometry (MS/MS) data for maize were collected from ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. These data are from a wide range of genetic backgrounds, including the inbred lines B73 and W22, many hybrids and their respective parents. Samples were collected from field trials, controlled environmental conditions, a range of (a)biotic conditions and different tissues, cell types and subcellular fractions. The protein search space included different maize genome annotations for the B73 inbred line from MaizeGDB, UniProtKB, NCBI RefSeq and for the W22 inbred line. 445 million MS/MS spectra were searched, of which 120 million were matched to 0.37 million distinct peptides. Peptides were matched to 66.2% of the proteins (one isoform per protein coding gene) in the most recent B73 nuclear genome annotation (v5). Furthermore, most conserved plastid- and mitochondrial-encoded proteins (NCBI RefSeq annotations) were identified. Peptides and proteins identified in the other searched B73 genome annotations will aid to improve maize genome annotation. We also illustrate high confidence detection of unique W22 proteins. N-terminal acetylation, phosphorylation, ubiquitination, and three lysine acylations (K-acetyl, K-malonyl, K-hydroxyisobutyryl) were identified and can be inspected through a PTM viewer in PeptideAtlas. All matched MS/MS-derived peptide data are linked to spectral, technical and biological metadata. This new PeptideAtlas is integrated with community resources including MaizeGDB at https://www.maizegdb.org/ and a peptide track in JBrowse. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/572651v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ec9005org.highwire.dtl.DTLVardef@1e35f34org.highwire.dtl.DTLVardef@7f5b2corg.highwire.dtl.DTLVardef@13acefd_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2023

Detection and editing of the updated plastid- and mitochondrial-encoded proteomes for Arabidopsis with PeptideAtlas

Arabidopsis thaliana Col-0 has plastid and mitochondrial genomes encoding for over one hundred proteins and several ORFs. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of such RNA editing at the protein level is largely unexplored due to the complexities of detection. This study first assembled the non-redundant set of identifiers, their correct protein sequences, and 452 predicted non-synonymous editing sites of which 56 are edited at lower frequency. Accumulation of edited and/or unedited proteoforms was then determined by searching [~]259 million raw MSMS spectra from ProteomeXchange as part of Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). All mitochondrial proteins and all except three plastid-encoded proteins (NDHG/NDH6, PSBM, RPS16), but none of the ORFs, were identified; we suggest that all ORFs and RPS16 are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical challenges of detection.167 predicted edit sites were detected at the proteome level. Minor frequency sites were indeed also edited at low frequency at the protein level. However, except for sites RPL5-22 and CCB382-124, proteins only accumulate in edited form (>98 -100% edited) even if RNA editing levels are well below 100%. This study establishes that RNA editing for major editing sites is required for stable protein accumulation.

plant biology↗

bioRxiv2023

Mapping the Arabidopsis thaliana proteome in PeptideAtlas and the nature of the unobserved (dark) proteome; strategies towards a complete proteome

This study describes a new release of the Arabidopsis thaliana PeptideAtlas proteomics resource providing protein sequence coverage, matched mass spectrometry (MS) spectra, selected PTMs, and metadata. 70 million MS/MS spectra were matched to the Araport11 annotation, identifying [~]0.6 million unique peptides and 18267 proteins at the highest confidence level and 3396 lower confidence proteins, together representing 78.6% of the predicted proteome. Additional identified proteins not predicted in Araport11 should be considered for building the next Arabidopsis genome annotation. This release identified 5198 phosphorylated proteins, 668 ubiquitinated proteins, 3050 N-terminally acetylated proteins and 864 lysine-acetylated proteins and mapped their PTM sites. MS support was lacking for 21.4% (5896 proteins) of the predicted Araport11 proteome - the dark proteome. This dark proteome is highly enriched for certain (e.g. CLE, CEP, IDA, PSY) but not other (e.g. THIONIN, CAP,) signaling peptides families, E3 ligases, TFs, and other proteins with unfavorable physicochemical properties. A machine learning model trained on RNA expression data and protein properties predicts the probability for proteins to be detected. The model aids in discovery of proteins with short-half life (e.g. SIG1,3 and ERF-VII TFs) and completing the proteome. PeptideAtlas is linked to TAIR, JBrowse, PPDB, SUBA, UniProtKB and Plant PTM Viewer.

plant biology↗

bioRxiv2021

The Arabidopsis thaliana PeptideAtlas; harnessing world-wide proteomics data for a comprehensive community proteomics resource

We developed a new resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis proteome, such as the significance of protein splice forms, post-translational modifications (PTMs), or simply obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) analyses collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical and biological metadata. Nearly 40 million out of [~]143 million MSMS spectra were matched to the reference genome Araport11, identifying [~]0.5 million unique peptides and 17858 uniquely identified proteins (only isoform per gene) at the highest confidence level (FDR 0.0004; 2 non-nested peptides [≥] 9 aa each), assigned canonical proteins, and 3543 lower confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified, generated from pseudogenes, alternative start, stops and/or splice variants and sORFs; these features should be considered for updates to the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. This new PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS data. One sentence summaryA new web resource providing the global community with mass spectrometry-based Arabidopsis proteome information and its spectral, technical and biological metadata integrated with TAIR and JBrowse

plant biology↗

bioRxiv2021

Tissue-type specific accumulation of the plastoglobular proteome, transcriptional networks and plastoglobular functions

Plastoglobules (PGs) are dynamic protein-lipid micro-compartments in plastids enriched for isoprenoid-derived metabolites. Chloroplast PGs support formation, remodeling and controlled dismantling of thylakoids during developmental transitions and environmental responses. However, the specific molecular functions of most PG proteins are still poorly understood. This study harnesses recent co-mRNA expression from ATTED-II using combined microarray and RNAseq information on an updated inventory of 34 PG proteins, as well as proteomics data across 30 Arabidopsis tissue types from ATHENA. Hierarchical clustering based on relative abundance for the PG proteins across non-photosynthetic and photosynthetic tissue types showed their coordinated protein accumulation across Arabidopsis parts, tissue types, development and senescence. We generated multiple mRNA-based networks by applying different coefficient thresholds; functional enrichment was determined for each network and PG gene. Combined analysis of these stringency networks identified a central hub and four peripheral modules. Enrichment of specific nuclear transcription factors (e.g. Golden2-like) and support for cross-talk between PGs and the plastid gene expression was observed, and specific ABC1 kinases seem part of a light signaling network. Examples of other specific findings are that FBN7b is involved with upstream steps of tetrapyrrole biosynthesis and that ABC1K9 is involved in starch metabolism. HighlightThe plastoglobular proteome shows coherent tissue-specific accumulation, whereas combined analysis of transcriptional co-expression networks, at different stringencies and following in-depth functional annotation, associate selected plastoglobular proteins to specific metabolic functions.

plant biology↗

bioRxiv2018

Extreme variation in rates of evolution in the plastid Clp protease complex

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multisubunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid-encoded gene (clpP1) and multiple nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid-encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find that clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g., loss of introns and RNA-editing sites) within seed plants. Although clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely the case. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genus Silene, which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear-encoded Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight correlation between amino-acid substitution rates in clpP1 and the nuclear-encoded Clp subunits across a broad sampling of angiosperms, suggesting ongoing selection on interactions within this complex.

evolutionary biology↗