Biology subjects

Goessweiner-Mohr, N.

Publications and source records attributed to Goessweiner-Mohr, N..

9 recordsLinked to original sources

bioRxiv2026

The plant specific VPS2.2/HYADE of the ESCRT-III protein sorting machinery localizes to a secretory compartment and is required for cell division plane determination in Arabidopsis

Positioning of the division plane is a critical step during cellular development. While in animal cells the plane of cell division is defined during metaphase, in plants this decision is made before mitosis and includes the formation of a cytoskeletal structure termed the preprophase band (PPB). Although not essential, the PPB constitutes the earliest mark for the plane of cell division and coincides with the sites where the cell plate fuses with the parental plasma membrane during cytokinesis. Recent studies indicate that endo- and exocytosis are involved in remodelling of the cortical division zone. Here we show that PPBs, phragmoplasts, cell plates and cell walls are misplaced in Arabidopsis mutants of Atvps2.2/hyade (hya), a plant-specific component of the Endosomal Sorting Complex Required for Transport (ESCRT)-III. We demonstrate that the amino acid substitution Q71P of the hya-3 allele abolishes the homo- and heteromerization with other ESCRT-III components. Functional HYA-GFP fusion proteins are excluded from the canonical location of ESCRT-III complexes at multivesicular bodies (MVBs) and are absent from Brefeldin A (BFA) and Wortmannin (WM) sensitive compartments in Arabidopsis. HYA-GFP neither localize to early nor late endosomes but colocalizes with markers of secretory compartments such as the Qc-SNARE, SYP61 and with trans-Golgi-network (TGN) derived secretory Rab-A3 vesicles. Moreover, HYA-GFP locates to the extracellular space indicating that VPS2.2/HYA is involved in secretion. These results are consistent with a novel, non-canonical function of ESCRT-III in the establishment and maintenance of the cortical division zone via secretion.

plant biology↗

bioRxiv2025

A conserved cytoplasmic glutamic acid mediates pH-dependent gating in bacterial urea channel UreI

The bacterial UreI channel family enables rapid urea uptake, essential for urease activity and acid resistance. While pH-dependent gating in Helicobacter pylori UreI (HpUreI) is attributed to periplasmic histidines, the role of cytoplasmic residues remains unexplored. The UreI homolog from Streptococcus salivarius (SsUreI), lacking periplasmic histidines, serves as a simplified model to identify gating determinants. Here, we combine yeast complementation assays, in vitro studies, and MD simulations to show that cytoplasmic glutamic acid E136 mediates pH-dependent gating in SsUreI. Mutagenesis in H. pylori/H. hepaticus homologs confirm E136 as a conserved pH sensor across UreI channels. Protonation of E136 disrupts its salt bridge with R20, increasing cytoplasmic-loop flexibility and urea-permeable filter conformations. These findings challenge the paradigm of exclusive periplasmic pH sensing, supporting a Gram-negative-specific dual-sensor model (E136 + histidines). By elucidating this molecular mechanism, we identify E136 as a therapeutic target to disrupt UreI-mediated acid resistance in pathogenic bacteria.

biophysics↗

bioRxiv2025

Clear Native Gel Electrophoresis for the Purification of Fluorescently Labeled Membrane Proteins in Native Nanodiscs

Native gel electrophoresis techniques, such as blue or clear native gel electrophoresis (BNE or CNE), are widely used to separate and characterize proteins. However, in high-resolution CNE, mild anionic or neutral detergents are often used at concentrations too low to prevent membrane-protein aggregation. Additionally, the identification of proteins is hampered by the lack of suitable molecular-weight markers, like those used in SDS-PAGE. Here, we introduce a novel approach that combines charged polymer-encapsulated nanodiscs and fluorescence correlation spectroscopy (FCS) to address both challenges. Membrane proteins are first extracted using Glyco-DIBMA, a negatively charged amphiphilic copolymer. This enables the spontaneous formation of nanodiscs harboring the fluorescently labeled target protein within a native-like lipid-bilayer environment, which is confirmed by FCS. The nanodiscs are then subjected to detergent-free CNE. As the number of protomers increases, the nanodiscs grow larger resulting in increased migration distances in CNE due to higher charge densities. Crucially, the nanodiscs remain intact throughout CNE, as demonstrated by FCS analysis of resolubilized bands excised from the gels. Moreover, the membrane proteins used in this study: a potassium channel (KvAP), a sodium channel (NavMs), a water channel (GlpF), and a urea channel (HpUreI) do not aggregate, as shown by the fluorescent brightnesses and diffusion times of individual nanodiscs. Moreover, the oligomeric states of membrane proteins can be deduced from the brightness per nanodisc. Since purified membrane proteins remain within a native-like lipid-bilayer environment and avoid detergent exposure, they are immediately suitable for downstream structural and functional studies.

biophysics↗

bioRxiv2025

Photomodulation of Kv Channel Activity

Voltage-gated potassium (Kv) channels shape action potentials, with their activity dependent on anionic lipids and modulated by membrane tension. However, the mechanism by which lipids regulate channel gating is not entirely clear. Cryo-electron microscopy studies suggest that in the down state-- typically associated with the inactive channel--arginines of the voltage sensor interact with lipid phosphates, pulling them upward and locally thinning the membrane by [~]5 [A]. This thinning is absent in the up state of the sensor, which is typically associated with the active channel. To test whether membrane thickness influences gating, we reconstituted Aeropyrum pernix Kv (KvAP) channels into planar lipid bilayers containing photoswitchable lipids. Blue light increased membrane thickness and KvAP activity, while UV light reversed these effects. Our results indicate that membrane thickening disrupts voltage sensor-lipid phosphate interactions, lowering the activation barrier for the up-movement of the voltage sensor and thus, channel opening. The resulting leftward shift in the current-voltage relationship demonstrates a light-driven approach to modulating Kv channel activity, with potential applications in neuronal excitability control. SignificanceVoltage-gated Kv channels respond to membrane voltage in a manner influenced by lipid composition and membrane tension. In the inactive state, voltage-sensing arginines interact with lipid phosphates, and when these interactions are strained--such as by membrane thickening or possibly by membrane tension--less voltage is required for channel opening. We demonstrate this interdependence by showing that a light-triggered increase in membrane thickness reversibly enhances Kv channel activity, directly linking membrane mechanics to voltage sensing. This work also establishes a light-based strategy for spatially precise Kv channel modulation.

biophysics↗

bioRxiv2023

YidC from Escherichia coli forms an ion-conducting pore upon activation by ribosomes.

The universally conserved protein YidC aids the insertion and folding of transmembrane polypeptides independently or as a part of the SecYEG translocon complex. In the former scenario, the lipid-exposed YidC surface equipped with a highly conserved positively charged arginine is thought to facilitate membrane insertion of the nascent chain by providing a countercharge for the acidic residues at the polypeptides N-terminal region. Here we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon binding a ribosome or ribosome-nascent chain complex (RNC). This pore is closed in the absence of ribosomes. As this pore is not visible in the published monomeric YidC structure, we used AlphaFold to construct the model of a parallel YidC dimer. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, and single-molecule fluorescence microscopy. In the dimeric model, the conserved positively charged arginine and many residues interacting with nascent chains point into the putative pore. This result suggests the possibility of an alternative mode of YidC-assisted membrane protein insertion.

biophysics↗

bioRxiv2023

Steady-state polypeptide transfer from the translocon to the membrane

In concert with irreversible non-equilibrium peptide translation by the ribosome, the nascent polypeptide chain may integrate into the membrane or translocate to the other side of the membrane, facilitated by the conserved protein translocation channel SecYEG in bacteria and Sec61 in eukaryotes. Assuming equilibrium for the decision processes yielded the biological hydrophobicity scale, reflecting free-energy differences {Delta}G between the pore interior and membrane. Yet kinetic effects and molecular dynamic simulations suggested that a nascent polypeptide could not sample the two separate environments a sufficient number of times for partitioning in equilibrium. Here we tested the hypothesis employing purified and reconstituted SecYEG harboring a stalled ribosome nascent chain (RNC). The SecYEG-RNC complex was open in a de-energized membrane, allowing ion flow. Application of a membrane potential closed the channel if nascent chain hydrophobicity permitted membrane integration. Taking the ratio of steady-state to initial ion conductances as a measure of nascent chain hydrophobicity, we found {Delta}G for KvAPs voltage sensor (4th helix harboring four arginines) and FtsQs transmembrane helix to be equal to 0.3 and -2.1 kcal/mol, respectively. Thus, {Delta}G observed in our minimalistic system agrees very well with the position-dependent amino acid contribution of the biological hydrophobicity scale. Characteristic sampling times of ~2 s appear sufficient to reach a steady state for a ~20 amino acid-long segment invalidating the hypothesis of insufficient sampling.

biophysics↗

bioRxiv2022

Trapped pore waters in the open proton channel HV1

The voltage-gated proton channel, HV1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, we estimate the water mobility for the putative case of an uninterrupted wire. The predicted single-channel water permeability 3x10-12cm3s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the prediction, i.e., it is negligible. Osmotic deflation of reconstituted lipid vesicles reveals trapped water inside the HV1 wild-type channel and D174A mutant open at 0 mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ~2 [A] capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance, conceivably by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while blocking water flow.

biophysics↗

bioRxiv2020

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

Many bacterial pathogens strictly rely on the activity of type III secretion systems (T3SSs) to secrete and translocate effector proteins in order to establish infection. The central component of T3SSs is the needle complex, a supramolecular machine which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to allow bacterial effectors to gain entry into the host cell cytoplasm to modulate signal transduction processes. Disruption of this process impairs pathogenicity, providing an avenue for antimicrobial design. However, the molecular principles underlying T3 secretion remain elusive. Here, we report the first structure of an active Salmonella enterica sv. Typhimurium needle complex engaged with the late effector protein SptP in two functional states, revealing the complete 800[A]-long secretion conduit and unravelling the critical role of the export apparatus (EA) subcomplex in T3 secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent transport, explaining heterogeneity and structural disorder of signal sequences in T3SS effector proteins. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates but prevents leaky pore formation to maintain the physical boundaries of compartments separated by a biological membrane. Following gate penetration, a moveable SpaR loop first folds up to then act akin to a linear ratchet to steer substrates through the needle complex. Together, these findings establish the molecular basis for substrate translocation through T3SSs, improving our understanding of bacterial pathogenicity and motility of flagellated bacteria, and paves the way for the development of novel concepts combating bacterial infections.

molecular biology↗

bioRxiv2019

Structural control for the coordinated assembly into functional pathogenic type-3 secretion systems

Functional injectisomes of the type-3 secretion system assemble into highly defined and stoichiometric bacterial molecular machines essential for infecting human and other eukaryotic cells. However, the mechanism that governs the regulated step-wise assembly process from the nucleation-phase, to ring-assembly, and the filamentous phase into a membrane embedded needle complex is unclear. We here report that the formation of a megadalton-sized needle complexes from Salmonella enterica serovar Typhimurium (SPI-1, Salmonella pathogenicity island-1) with proper stoichiometries is highly structurally controlled competing against the self-assembly propensity of injectisome components, leading to a highly unusual structurally-pleiotropic phenotype. The structure of the entire needle complex from pathogenic injectisomes was solved by cryo electron microscopy, focused refinements (2.5-4 [A]) and co-variation analysis revealing an overall asymmetric arrangement containing cyclic, helical, and asymmetric sub-structures. The centrally located export apparatus assembles into a conical, pseudo-helical structure and provides a structural template that guides the formation of a 24-mer cyclic, surrounding ring, which then serves as a docking interface comprising three different conformations for sixteen N-terminal InvG subunits of the outer secretin ring. Unexpectedly, the secretin ring excludes the 16th protein chain at the C-terminal outer ring, resulting in a pleiotropic 16/15-mer ring and consequently to an overall 24:16/15 basal body structure. Finally, we report how the transition from the pseudo-helical export apparatus into the helical filament is structurally resolved to generate the protein secretion channel, which provides the structural basis to restrict access of unfolded effector substrates. These results highlight the diverse molecular signatures required for a highly coordinated assembly process and provide the molecular basis for understanding triggering and transport of unfolded proteins through injectisomes.

molecular biology↗