Diffusion and localization of proteins in the plasma membrane of Saccharomyces cerevisiae

Yeast (Saccharomyces cerevisiae) plasma membrane is unique, as diffusion is 3-4 orders of magnitude slower than in other known membranes. In this thesis, we study diffusion and localization of proteins in the yeast plasma membrane. In the yeast plasma membrane, proteins are not uniformly distributed, many of them show localization to distinct domains. MCC/eisosomes are one type of such domains. They are very stable compartments that do not change for longer periods of time than cell cycle of the yeast cells. We have developed a new method of immobilization of the cells allowing us to study them in more detail. Using that method, we looked at the diffusion and localization of proteins in relation to the MCC/eisosomes. Some proteins (like Can1p) are immobilized in the MCC/eisosomes, can diffuse through the MCC/eisosomes without immobilization (like Nha1), or are excluded from them (like Pma1p). However, the structure does not have an effect on diffusion coefficients of any of the groups. Additionally, we have discovered, that proteins can be excluded from the MCC/eisosomes due to large cytoplasmic domains close to the plasma membrane. We also tried to determine the cause(s) of the slow diffusion in the yeast plasma membrane, and while we think lipid composition of the membrane has major impact on it, we are still far from any conclusions

A trifunctional linker for palmitoylation and peptide and protein localization in biological membranes

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

Disaccharides impact the lateral organization of lipid membranes

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