RÉSUMÉ
The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell.
Sujet(s)
Protéines Escherichia coli , Escherichia coli , Pression osmotique , Escherichia coli/métabolisme , Escherichia coli/génétique , Protéines Escherichia coli/métabolisme , Protéines Escherichia coli/composition chimique , Protéines Escherichia coli/génétique , Modèles moléculaires , Conformation des protéines , Cryomicroscopie électronique , Spectroscopie par résonance magnétique , Domaines protéiquesRÉSUMÉ
Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (Lo ) and liquid-disordered (Ld ) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane-spanning peptide.
Sujet(s)
Membrane cellulaire/métabolisme , Double couche lipidique/métabolisme , Microdomaines membranaires/métabolisme , Acide palmitique/composition chimique , Fragments peptidiques/métabolisme , Protéines/métabolisme , Liposomes unilamellaires/métabolisme , Membrane cellulaire/composition chimique , Humains , Double couche lipidique/composition chimique , Lipoylation , Microdomaines membranaires/composition chimique , Fragments peptidiques/composition chimique , Maturation post-traductionnelle des protéines , Protéines/composition chimique , Tyrosine/composition chimique , Tyrosine/métabolisme , Liposomes unilamellaires/composition chimiqueRÉSUMÉ
We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to â¼2.5 h.
Sujet(s)
Perméabilité des membranes cellulaires , Liposomes/métabolisme , Saccharomyces cerevisiae/cytologie , Concentration en ions d'hydrogène , Cinétique , Spectrométrie de fluorescenceRÉSUMÉ
Super-resolution imaging and single-particle tracking require cells to be immobile as any movement reduces the resolution of the measurements. Here, we present a method based on APTES-glutaraldehyde coating of glass surfaces to immobilize cells without compromising their growth. Our method of immobilization is compatible with Saccharomyces cerevisiae, Escherichia coli, and synthetic cells (here, giant-unilamellar vesicles). The method introduces minimal background fluorescence and is suitable for imaging of single particles at high resolution. With S. cerevisiae we benchmarked the method against the commonly used concanavalin A approach. We show by total internal reflection fluorescence microscopy that modifying surfaces with ConA introduces artifacts close to the glass surface, which are not present when immobilizing with the APTES-glutaraldehyde method. We demonstrate validity of the method by measuring the diffusion of membrane proteins in yeast with single-particle tracking and of lipids in giant-unilamellar vesicles with fluorescence recovery after photobleaching. Importantly, the physical properties and shape of the fragile GUVs are not affected upon binding to APTES-glutaraldehyde coated glass. The APTES-glutaraldehyde is a generic method of immobilization that should work with any cell or synthetic system that has primary amines on the surface.
Sujet(s)
Cellules artificielles/métabolisme , Escherichia coli/métabolisme , Glutaraldéhyde/pharmacologie , Microscopie de fluorescence/méthodes , Saccharomyces cerevisiae/métabolisme , Liposomes unilamellaires/métabolisme , Concanavaline A/pharmacologie , Escherichia coli/croissance et développement , Imagerie optique/méthodes , Saccharomyces cerevisiae/croissance et développement , Propriétés de surface/effets des médicaments et des substances chimiquesRÉSUMÉ
The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.
Sujet(s)
Systèmes de transport d'acides aminés basiques/composition chimique , Membrane cellulaire/métabolisme , Proton-Translocating ATPases/composition chimique , Protéines de Saccharomyces cerevisiae/composition chimique , Saccharomyces cerevisiae/métabolisme , Systèmes de transport d'acides aminés basiques/métabolisme , Membrane cellulaire/ultrastructure , Diffusion , Redistribution de fluorescence après photoblanchiment , Cinétique , Microdomaines membranaires , Conformation des protéines , Transport des protéines , Proton-Translocating ATPases/métabolisme , Saccharomyces cerevisiae/ultrastructure , Protéines de Saccharomyces cerevisiae/métabolismeRÉSUMÉ
Disaccharides are well-known for their membrane protective ability. Interaction between sugars and multicomponent membranes, however, remains largely unexplored. Here, we combine molecular dynamics simulations and fluorescence microscopy to study the effect of mono- and disaccharides on membranes that phase separate into Lo and Ld domains. We find that nonreducing disaccharides, sucrose and trehalose, strongly destabilize the phase separation leading to uniformly mixed membranes as opposed to monosaccharides and reducing disaccharides. To unveil the driving force for this process, simulations were performed in which the sugar linkage was artificially modified. The availability of accessible interfacial binding sites that can accommodate the nonreducing disaccharides is key for their strong impact on lateral membrane organization. These exclusive interactions between the nonreducing sugars and the membranes may rationalize why organisms such as yeasts, tardigrades, nematodes, bacteria, and plants accumulate sucrose and trehalose, offering cell protection under anhydrobiotic conditions. The proposed mechanism might prove to be a more generic way by which surface bound agents could affect membranes.