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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 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, eLife digest Cells often communicate with each other by releasing chemicals that normally are stored in small membrane-bound compartments called vesicles. For example, when a neuron is stimulated, vesicles merge with its cell membrane and release their content into a gap between itself and other neurons. This complicated process involves many steps and molecules, including proteins called SNAREs. Some SNARE proteins reside at the inner side of the cell membrane and help vesicles to fuse with this membrane. Two SNARE proteins called SNAP25 and SNAP23 are produced in the liquid inside the cell and initially float freely. Eventually, these proteins become directly anchored to the cell membrane, however, not much is known about what happens to these proteins in between these stages, or how they first attach to the membrane before anchoring to it. Electrostatic forces between oppositely charged molecules are known to be important for them to bind with each other. Here, electrostatic forces are less likely to occur because SNAP25 and SNAP23 are both mostly negatively charged, and should therefore be repelled from the cell membrane, which also typically has a negative charge. However, both SNAP25 and SNAP23 have a small cluster of positively charged amino acids (the building blocks of proteins) near the attachment site, and Weber et al. have now tested whether this charge is sufficient to overcome the predicted repulsion. The approach involved making mutant proteins with either more or less positively charged attachment regions. Mutant SNAP25 or SNAP23 proteins with more positive charges may stick more tightly but not necessarily more permanently to the membrane. However, when the number of positive charges was lowered, more of the proteins remained floating freely in the liquid inside the cell. These results suggest that even a small number of positively charged amino acids is sufficient to help a protein bind to a cell membrane for further processing. The findings of Weber et al. reveal an early step in the life cycle of SNAP25 and SNAP23 before they anchor to the cell membrane. They suggest that finely tuned protein electrostatics can regulate how long a protein spends at a specific site and thereby indirectly determine its fate. Such fine-tuned protein electrostatics are difficult to recognize and could represent an underestimated regulatory mechanism in all types of cells. DOI: http://dx.doi.org/10.7554/eLife.19394.002