The packing density of a supramolecular membrane protein cluster is controlled by cytoplasmic interactions.

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.

No Evidence for Spontaneous Lipid Transfer at ER-PM Membrane Contact Sites.

Non-vesicular lipid transport steps play a crucial role in lipid trafficking and potentially include spontaneous exchange. Since membrane contact facilitates this lipid transfer, it is most likely to occur at membrane contact sites (MCS). However, to date it is unknown whether closely attached biological membranes exchange lipids spontaneously. We have set up a system for studying the exchange of lipids at MCS formed between the endoplasmic reticulum (ER) and the plasma membrane. Contact sites were stably anchored and the lipids cholesterol and phosphatidylcholine (PC) were not capable of transferring spontaneously into the opposed bilayer. We conclude that physical contact between two associated biological membranes is not sufficient for transfer of the lipids PC and cholesterol.

Intramolecular interactions within the human immunodeficiency virus-1 gp41 loop region and their involvement in lipid merging.

The human immunodeficiency virus utilizes its gp41 fusion protein to mediate virus-cell membrane fusion. The conserved disulfide loop region in the gp41 hairpin conformation reverses the protein chain, such that the N-terminal heptad repeat and the C-terminal heptad repeat regions interact to form the six-helix bundle. Hence, it is conceivable that the sequential folded N- and C-terminal parts of the loop region also interact. We show that the N- and C-terminal parts of the loop preferably form disulfide-bonded heterodimers with slow oxidation kinetics. Furthermore, when the two parts were linked to a single polypeptide to form the full-length loop, only an intramolecular disulfide-bonded loop was formed. Fluorescence studies revealed that an interaction takes place between the N- and C-terminal parts of the loop in solution, which was sustained in membranes. Functionally, only a combination of the N- and C-loop parts induced lipid mixing of model liposomes, the level of which increased 8-fold when they were connected to a single polypeptide chain. In both cases, the activity was independent of the oxidation state of the cysteines. Overall, the data (i) provide evidence of a specific interaction between the N- and C-terminal parts of the loop, which can further stabilize gp41 hairpin conformation, and (ii) suggest that the interaction between the N- and C-terminal parts of the loop is sufficient to induce lipid merging without forming a disulfide bond.

Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors.

The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an α-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide's affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.