An Stomatin, Prohibitin, Flotillin, and HflK/C-Domain Protein Required to Link the Phage-Shock Protein to the Membrane in Bacillus subtilis

Membrane surveillance and repair is of utmost importance to maintain cellular integrity and allow cellular life. Several systems detect cell envelope stress caused by antimicrobial compounds and abiotic stresses such as solvents, pH-changes and temperature in bacteria. Proteins containing an Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain, including bacterial flotillins have been shown to be involved in membrane protection and membrane fluidity regulation. Here, we characterize a bacterial SPFH-domain protein, YdjI that is part of a stress induced complex in Bacillus subtilis. We show that YdjI is required to localize the ESCRT-III homolog PspA to the membrane with the help of two membrane integral proteins, YdjG/H. In contrast to classical flotillins, YdjI resides in fluid membrane regions and does not enrich in detergent resistant membrane fractions. However, similarly to FloA and FloT from B. subtilis, deletion of YdjI decreases membrane fluidity. Our data reveal a hardwired connection between phage shock response and SPFH proteins.

Detergent Resistant Membrane Domains in Broccoli Plasma Membrane Associated to the Response to Salinity Stress

Detergent-resistant membranes (DRMs) microdomains, or “raft lipids”, are key components of the plasma membrane (PM), being involved in membrane trafficking, signal transduction, cell wall metabolism or endocytosis. Proteins imbibed in these domains play important roles in these cellular functions, but there are few studies concerning DRMs under abiotic stress. In this work, we determine DRMs from the PM of broccoli roots, the lipid and protein content, the vesicles structure, their water osmotic permeability and a proteomic characterization focused mainly in aquaporin isoforms under salinity (80 mM NaCl). Based on biochemical lipid composition, higher fatty acid saturation and enriched sterol content under stress resulted in membranes, which decreased osmotic water permeability with regard to other PM vesicles, but this permeability was maintained under control and saline conditions; this maintenance may be related to a lower amount of total PIP1 and PIP2. Selective aquaporin isoforms related to the stress response such as PIP1;2 and PIP2;7 were found in DRMs and this protein partitioning may act as a mechanism to regulate aquaporins involved in the response to salt stress. Other proteins related to protein synthesis, metabolism and energy were identified in DRMs independently of the treatment, indicating their preference to organize in DMRs.

Phase Diagram of Purified CNS Myelin Reveals Continuous Transformation between Expanded and Compacted Lamellar States

Purified myelin membranes (PMMs) are the starting material for biochemical studies, from individual components up to the isolation of detergent-resistant membrane (DRM) fractions or detergent-insoluble glycosphingolipid (DIG) fractions, which are commonly believed to resemble physiological lipid rafts. The normal DIG isolation protocol involves the extraction of lipids under moderate cooling. The isolation of PMMs also involves the cooling of myelin as well as exposure to low ionic strength (IS). Here, we addressed the combined influence of cooling and IS on the structure of PMMs. The phase behaviour was investigated by small angle X-ray diffraction. Analysis of the diffraction peaks revealed the lamellar periodicity ( d ), the number of periodically correlated bilayers ( N ), and the relatives fractions of each phase. Departure from physiological conditions induced a phase separation in myelin. The effect of monovalent and divalent ions was also compared at equivalent IS, showing a differential effect, and phase diagrams for both ion types were established—Ca2+ induced the well-known over-compacted phase, but additionally we also found an expanded phase at low IS. Na+ promoted phase separation, and also induced over-compaction at sufficiently high IS. Finally, exploring the whole phase diagram, we found evidence for the direct isothermal transformation from the expanded to the compacted phase, suggesting that both phases could in fact originate from the identical primary lateral phase separation, whereas the apparent difference lies in the inter-bilayer interaction that is modulated by the ionic milieu.

Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.

Detergent-resistant microdomains (lipid rafts) in endomembranes of the wild halophytes

In the present work, we studied detergent-resistant membrane microdomains (DRM) of chloroplasts and mitochondria – organelles that provide photosynthesis and respiration in a plant cell. The objects of the study were euhalophyte Salicorniaperennans Willd., which relates to salt-accumulating plants and glycohalophyte Artemisia santonica L., which relates to salt-excluder plants. To get DRM, the chloroplast and mitochondria fractions were solubilised with a solution containing Triton X-100. The resulting material was introduced in sucrose gradient of 35–25–15–5% and centrifuged at 200000 g, 2 h. The presence of an opalescent detergent-resistant zone of membranes in 15% sucrose layer and a specific lipid composition of this zone were the signs of successful rafts obtaining of. The isolated DRM are sterol- and cerebroside-enriched (27–89% of the sum of membrane lipids) domains with a high degree of saturation of fatty acids composition (more than 50% of the sum). The main DRM-specific lipids of chloroplast of A. santonica glycohalophyte are cerebrosides, whereas those of S. perennans euhalophyte are sterols. The revealed differences in the composition of raft-forming lipids in chloroplast and mitochondria halophyte membranes, differing in the salt-resistance strategy, suggest the participation of rafts in salt-resistance mechanisms.

Membrane-Bound Class III Peroxidases: Unexpected Enzymes with Exciting Functions

Class III peroxidases are heme-containing proteins of the secretory pathway with a high redundance and versatile functions. Many soluble peroxidases have been characterized in great detail, whereas only a few studies exist on membrane-bound isoenzymes. Membrane localization of class III peroxidases has been demonstrated for tonoplast, plasma membrane and detergent resistant membrane fractions of different plant species. In silico analysis revealed transmembrane domains for about half of the class III peroxidases that are encoded by the maize (Zea mays) genome. Similar results have been found for other species like thale-cress (Arabidopsis thaliana), barrel medic (Medicago truncatula) and rice (Oryza sativa). Besides this, soluble peroxidases interact with tonoplast and plasma membranes by protein–protein interaction. The topology, spatiotemporal organization, molecular and biological functions of membrane-bound class III peroxidases are discussed. Besides a function in membrane protection and/or membrane repair, additional functions have been supported by experimental data and phylogenetics.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment

Background Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored. Objective This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains. Methods Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined. Results Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density (ρ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI. Conclusion Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity.