No abstract

Molecule clustering is an important mechanism underlying cellular self-organization. In the cell membrane, a variety of fundamentally different mechanisms drive membrane protein clustering into nanometre-sized assemblies. To date, it is unknown whether this clustering process can be dissected into steps differentially regulated by independent mechanisms. Using clustered syntaxin molecules as an example, we study the influence of a cytoplasmic protein domain on the clustering behaviour. Analysing protein mobility, cluster size and accessibility to myc-epitopes we show that forces acting on the transmembrane segment produce loose clusters, while cytoplasmic protein interactions mediate a tightly packed state. We conclude that the data identify a hierarchy in membrane protein clustering likely being a paradigm for many cellular self-organization processes.DOI: http://dx.doi.org/10.7554/eLife.20705.001

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.DOI: http://dx.doi.org/10.7554/eLife.19394.001

No abstract

Molecule clustering is an important mechanism underlying cellular self-organization. In the cell membrane, a variety of fundamentally different mechanisms drive membrane protein clustering into nanometre-sized assemblies. To date, it is unknown whether this clustering process can be dissected into steps differentially regulated by independent mechanisms. Using clustered syntaxin molecules as an example, we study the influence of a cytoplasmic protein domain on the clustering behaviour. Analysing protein mobility, cluster size and accessibility to myc-epitopes we show that forces acting on the transmembrane segment produce loose clusters, while cytoplasmic protein interactions mediate a tightly packed state. We conclude that the data identify a hierarchy in membrane protein clustering likely being a paradigm for many cellular self-organization processes.