Biology subjects

Exterkate, M.

Publications and source records attributed to Exterkate, M..

4 recordsLinked to original sources

bioRxiv2026

The multidrug transporter Pdr5 from Saccharomyces cerevisiae is an ergosterol-modulated efflux pump

The pleiotropic drug resistance ABC transporter Pdr5 and its homologs play a critical role in multidrug resistance of numerous fungi, including pathogens. Previous studies demonstrated that alterations in the composition of the plasma membrane in which Pdr5 is embedded, specifically the ergosterol content, affected transport activity. Here we report the cryo-EM map of Saccharomyces cerevisiae Pdr5 in complex with ATP/ADP at 2.2 [A] resolution, revealing densities on the surface of the transmembrane domains as well as inside the substrate-binding site, that were assigned as ergosterol, identified and quantified by GC-MS, and the sphingolipid M(IP)2C, identified via native MS. The ergosterol molecules are bound to the protein in well-defined grooves and unexpected locations. We found that the activity of Pdr5 follows a Gaussian-like distribution, increasing with the ergosterol-concentration to reach a maximum before declining at higher concentrations, but this modulation was not observed in the presence of cholesterol, the sterol analogue found in mammalian cells. Mutational analysis corroborates the role of a single ergosterol-binding site in the modulation of Pdr5 activity. Our structural analysis identifies a protein-lipid network that could serve as a center for lipid engagement linking the surrounding lipid environment to the substrate-binding region of Pdr5. These findings advance our understanding of Pdr5 function by demonstrating that the lipid environment is closely linked to Pdr5 structure and activity.

biochemistry↗

bioRxiv2026

Control of motility and cell shape of Haloferax volcanii is linked by a transcriptional regulator

Archaea rely on motility and morphological plasticity to navigate their environments, yet the transcriptional regulation of these processes remains poorly understood. In Haloferax volcanii, archaellum-dependent motility is transcriptionally regulated, but an EarA-like master regulator is absent. Here, we identify CsmR as a transcriptional regulator that links archaellum biogenesis and cell-shape transitions in H. volcanii. Deletion of csmR abolished detectable motility, whereas overexpression increased motility and promoted a sustained rod-like morphology. Comparative transcriptomics defined a CsmR-associated regulon that includes archaellum and chemotaxis genes as well as rod-shape determinants (e.g., Sph3 and RdfA), and upstream motif enrichment supports a direct role for CsmR in transcriptional control. Furthermore, csmR and cirA, a KaiC-like regulator, share extensive transcriptional overlap, with CirA likely fine-tuning CsmR-mediated regulation through post-translational modification. These findings establish CsmR as a key integrator of motility and cell shape regulation in Haloferax volcanii, suggesting that haloarchaea coordinate these fundamental processes through an unidentified transcriptional network. Moreover, Northern blotting and cell shape observation suggest that transcription factor RosR is involved in the regulation of an sRNA that shares extensive overlap with the cirA gene, possibly fine-tuning the effect of CirA on the regulation of the archaellum cluster and the rod shape determinants sph3 and rdfA. Understanding this interplay provides new insights into archaeal adaptability and may reveal broader regulatory principles in prokaryotic cell biology.

microbiology↗

bioRxiv2025

Acylation of the RTX Toxin MbxA stimulates host membrane disruption through a specific interaction with cholesterol

RTX toxins, characterized by their calcium-binding glycine-rich repeat regions, are known for their ability to disrupt host cell membranes. Although the acylation of specific lysine residues in these toxins is established as crucial for their hemolytic activity, the precise mechanisms underlying this enhancement remain unclear. In this study, we explore the role of acylation in the pore-forming behaviour of the RTX toxin MbxA by comparing the lytic activities of acylated MbxA and its non-acylated form, proMbxA, on in vitro membrane systems, as well as living cells. Our findings demonstrate that a cholesterol specific interaction promotes MbxA- induced membrane disruption in an acylation-dependent manner. Further analysis revealed that acylation is not necessary for initial membrane binding but markedly enhances pore formation. Overall, our results offer valuable insights into the molecular determinants that regulate RTX toxin activity, highlighting a complex interplay between lipid composition (sterols), acylation, and membrane disruption, thereby advancing our understanding of RTX toxin pathogenesis.

biochemistry↗

bioRxiv2020

A promiscuous archaeal cardiolipin synthase generating a variety of cardiolipins and phospholipids

Cardiolipin (DPCL) biosynthesis has barely been explored in Archaeal isoprenoid-based ether lipid membranes. Here, we identified a cardiolipin synthase (MhCls) from the mesophilic anaerobic methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and subsequently characterized by LC-MS. MhCls utilizes two archaetidylglycerol molecules in a transesterification reaction to synthesize archaeal di-phosphate cardiolipin (aDPCL) and glycerol. The enzyme is invariant to the stereochemistry of the glycerol-backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol to generate di-phosphate cardiolipin (DPCL). Remarkably, in the presence of archaetidylglycerol and phosphatidylglycerol, MhCls formed an archaeal-bacterial hybrid di-phosphate cardiolipin (hDPCL), that so far has not been observed in nature. Due to the reversibility of the transesterification, cardiolipin can be converted back in presence of glycerol into phosphatidylglycerol. In the presence of other compounds that contain primary hydroxyl groups (e.g. alcohols, water, sugars) various natural and unique artificial phospholipid species could be synthesized, including multiple di-phosphate cardiolipin species. Moreover, MhCls could utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphate cardiolipin, emphasizing the promiscuity of this cardiolipin synthase.

molecular biology↗