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.

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 $2.5 h.

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.

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.

The structure of a trifunctional linker is shown. The linker contains a fluorescent dye (green), two palmitoyl moieties (light blue) and is covalently coupled to a protein (schematically shown in dark blue). The tripartite is used to study the effect palmitoylation on the localization of membrane proteins or peptides in giant‐unilamellar vesicles (GUVs) composed of a phase‐separating lipid mixture. Here, the palmitoylated protein is shown to localize to the liquid‐disordered part of the membrane (thinner membrane) as shown by the co‐localization with a specific lipid probe (red). More information can be found in the full paper by G. Roelfes, B. Poolman et al. on page 1320 in Issue 9, 2020 (DOI: 10.1002/cbic.201900655).