Biology subjects

Wunder, T.

Publications and source records attributed to Wunder, T..

5 recordsLinked to original sources

bioRxiv2026

The Conserved N-Terminal Extension of AtKEA1 Is Largely Dispensable for Plastid Function but Contributes to Potassium Homeostasis

Members of the K efflux antiporter (KEA) family fulfill key roles in plastids and the endomembrane system. Plants and green algae possess at least one KEA mediating K/H exchange across the plastid inner envelope (IE) membrane. Recently, IE KEAs were shown to be essential for plastid gene expression (PGE), chloroplast development, and photosynthesis. Plants lacking these antiporters exhibit reduced stromal protein synthesis and accumulation of unprocessed rRNA precursors. KEA proteins comprise a conserved monovalent cation/proton antiporter 2 (CPA2) domain and a regulatory K transport and NAD-binding (KTN) domain. IE KEAs are distinguished by an additional [~]500-amino-acid N-terminal extension containing a coiled-coil (CC) domain embedded within a largely intrinsically disordered region (IDR). Intrigued by this unusual architecture, we performed phylogenetic analyses, revealing that this N-terminal fusion arose early and has been conserved throughout the green lineage. We then investigated the oligomeric state, native distribution, and function of the N-terminal domain. Using Arabidopsis thaliana, we found that IE KEAs localize to discrete clusters within the inner envelope membrane and assemble into complexes of approximately 600 kDa. Finally, complementary approaches using a functional KEA1 variant lacking the core N-terminal domains (KEA1{Delta}N) indicate that this extension plays a regulatory rather than an essential role. Our findings uncover an evolutionarily ancient regulatory module that shapes the molecular organization and function of IE KEAs, advancing our understanding of plastid ion and pH homeostasis and plastid ribosome integrity. One-sentence summaryPlastid KEA1/2 proteins feature a unique N-terminal extension that modulates potassium transport activity in a yet unknown manner but is not essential for normal plant growth under ambient conditions.

plant biology↗

bioRxiv2026

A nucleotide-triggered molecular switch orchestrating septin polymerization

Septins are conserved cytoskeletal GTP-binding proteins that form higher-order structures critical for cytokinesis and polarized growth across opisthokonts. In budding yeast and humans, four homologous septins assemble into hetero-octameric protofilaments through alternating interactions between adjacent G-domains (G-interface) or N- and C-termini (NC-interface), which then polymerize end-to-end into filaments. How nucleotide binding and hydrolysis control filament assembly has remained elusive. We uncover a septin-specific mechanism in which nucleotide binding stabilizes the monomeric G-interface and primes it for protofilament formation. Cryo-EM and DEER spectroscopy reveal nucleotide-induced conformational changes that engage the G-interface and enable an NC-compatible conformation absent in the nucleotide-free state. In vitro binding and reconstitution assays confirm that G-interface formation is strictly nucleotide-dependent and required for subsequent NC-interface assembly. These findings establish unidirectional allosteric signaling from the G-to the NC-interface, revealing the molecular basis for controlled septin protofilament assembly.

molecular biology↗

bioRxiv2025

Conserved and Lineage-Specific Roles of KEA-Mediated Ion Homeostasis in Chlamydomonas

In Arabidopsis thaliana, seamless plastid gene expression and development depend on finely balanced ion homeostasis across the inner envelope (IE) membrane, maintained by the K/H antiporters AtKEA1/2. To assess whether these functions are retained across mono- and polyplastidic representatives of the green lineage, we studied CrKEA1, the sole IE KEA homolog in the unicellular alga Chlamydomonas reinhardtii. Using CRISPR/Cas9, we generated a Cr-kea1 knockout mutant that exhibits impaired photoautotrophic growth, chloroplast deformation, and photoinhibition. Transcriptomics revealed strong induction of ribosome biogenesis genes and reduced abundance of transcripts associated with cell and plastid division. Further RNA analyses confirmed defects in stromal rRNA maturation of Cr-kea1, paralleling observations from Arabidopsis kea1kea2 mutants. Expression of CrKEA1 in Arabidopsis rescued growth and rRNA maturation in At-kea1kea2, demonstrating functional continuity after the ancient divergence between the two lineages. Cross-species transcriptomic comparisons further revealed that IE KEA loss elicits both shared and species-specific transcriptional responses: PhANG repression was conserved between algae and plants, whereas activation of the chloroplast unfolded protein response (cpUPR) and reduced expression of genes tied to cell-cycle and plastid fission occurred only in Chlamydomonas. Single-cell time-lapse imaging confirmed that Cr-kea1 exhibits an increased frequency of aberrant cytokinesis, unequal division, and division failure. Our findings demonstrate that while IE KEA transporters fulfill conserved roles in maintaining the conditions for plastid gene expression, their integration into broader cellular networks has diverged between unicellular chlorophytes and embryophytes (land plants). This underscores a lineage-dependent tuning of plastid-nucleus communication shaped by organismal complexity and plastid number. One-sentence summaryDisruption of KEA-mediated chloroplast ion homeostasis in Chlamydomonas reinhardtii reveals conserved and lineage-specific control of plastid rRNA processing and cell cycle progression.

plant biology↗

bioRxiv2025

Catch & Release - rapid cost-effective protein purification from plants using a DIY GFP-Trap-protease approach

The purification of proteins is the foundation to study their structure, function, biochemical properties, and interaction partners. In plant research, unique challenges arise from the complexity of plant tissues, interference of secondary metabolites, and sometimes the low abundance of target proteins. Many conventional plant protein purification methods rely on expensive reagents, multi-step procedures, and labor-intensive workflows, limiting their feasibility for many applications. Here, we present the "Catch & Release" system, a cost-effective, fast and reliable one-step purification workflow for the isolation of soluble and membrane-bound proteins from plant tissues. The Catch & Release toolbox includes a vector set, a homemade GFP-trap and homemade proteases. Catch & Release vectors streamline cloning and transgenic plant selection through the Fluorescence-Accumulating Seed Technology (FAST), which marks positive transformants with a strongly fluorescing seed coat. Each plasmid consists of four, easy to exchange, modules: a plant promoter, a cloning dropout marker, protease cleavage sites, and seven different epitope tags, including an innovative dual-fluorescent tag, providing flexibility for diverse experimental needs. The in vivo functionality of all modules has been confirmed. Besides enabling standard molecular biological experimentation, our vector set in combination with homemade GFP-trap and proteases enables efficient and rapid isolation of soluble and high molecular weight membrane proteins directly from plants. By following our detailed reagent preparation instructions, purification costs can be decreased hundred-fold compared to the commercially available options.

plant biology↗

bioRxiv2020

The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

molecular biology↗