Driving a planar model system into the 3(rd) dimension: generation and control of curved pore-spanning membrane arrays.

The generation of a regular array of micrometre-sized pore-spanning membranes that protrude from the underlying surface as a function of osmotic pressure is reported. Giant unilamellar vesicles are spread onto non-functionalized Si/SiO(2) substrates containing a highly ordered array of cavities with pore diameters of 850 nm, an interpore distance of 4 µm and a pore depth of 10 µm. The shape of the resulting pore-spanning membranes is controlled by applying an osmotic pressure difference between the bulk solution and the femtoliter-sized cavity underneath each membrane. By applying Young-Laplace's law assuming moderate lateral membrane tensions, the response of the membranes to the osmotic pressure difference can be theoretically well described. Protruded pore-spanning membranes containing the receptor lipid PIP(2) specifically bind the ENTH domain of epsin resulting in an enlargement of the protrusions and disappearance as a result of ENTH-domain induced defects in the membranes. These results are discussed in the context of an ENTH-domain induced reduction of lateral membrane tension and formation of defects as a result of helix insertion of the protein in the bilayer.

The mitochondrial oxidase assembly protein1 (Oxa1) insertase forms a membrane pore in lipid bilayers.

The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.