Sorting of Streptavidin Protein Coats on Phase-Separating Model Membranes

Abstract

Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin

“…S4). The tetravalent binding of streptavidin to DPPEbiotin yielded no apparent variations in the membrane topography as assessed with pPLM at any of the streptavidin or DPPE-biotin concentrations tested, consistent with previous reports (26). Taken together, these findings support the hypothesis that the spatial organization of GM1 binding sites in CTxB is important for the curvature generation process.…”

Adhesion Stabilizes Robust Lipid Heterogeneity in Supercritical Membranes at Physiological Temperature

“…This work is consistent with earlier studies in suggesting that lipid-mediated membrane heterogeneity can be stabilized by controlling the localization of a subset of components. Previous work in model membranes with coexisting L o and L d phases have shown that lipid heterogeneity is stabi-lized when lipids are tethered to cross-linked actin meshworks (39) or to 2D crystals of avidin (40). In both cases, experimental results of probe partitioning into or away from protein-coated membrane domains was interpreted as an increase in transition temperature.…”

Physical Aspects of Viral Membrane Fusion

“…By using streptavidin-coated surfaces, individual biotinylated vesicles can be immobilized on surfaces for imaging [116,117]. The presence of the surface may affect the free energy of the system [118], but the ability to make extended observations of a single virus-liposome complex provides an approach that can overcome the limitations of bulk fusion assays due to the stochastic nature of each fusion event and the difficulty in synchronizing fusion of large ensembles of particles. Liposome targets also allow the role of membrane curvature to be tested in fusion experiments.…”