Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase.

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

An alliance between lipid transfer proteins and scramblases for membrane expansion.

Membrane growth requires lipid supply, which is usually accomplished by lipid synthesis or vesicular trafficking. In the case of autophagosomes, these principles do not apply. Ghanbarpour et al. postulate that autophagosome expansion relies on non-vesicular lipid delivery from the ER, whereby the activity of a lipid transfer protein (LTP) is directly coupled to scramblase activities in the donor and acceptor bilayers1. This new concept opens the possibility that lipid traffic is controlled by scramblases that provide not only specific docking sites for LTPs, thereby directing lipid flow, but also support their activity by overcoming barriers for lipid extraction and deposition.

Factors influencing the solubilization of membrane proteins from Escherichia coli membranes by styrene-maleic acid copolymers.

Styrene-maleic acid (SMA) copolymers are a promising alternative to detergents for the solubilization of membrane proteins. Here we employ Escherichia coli membranes containing KcsA as a model protein to investigate the influence of different environmental conditions on SMA solubilization efficiency. We show that SMA concentration, temperature, incubation time, ionic strength, presence of divalent cations and pH all influence the amount of protein that is extracted by SMA. The observed effects are consistent with observations from lipid-only model membrane systems, with the exception of the effect of pH. Increasing pH from 7 to 9 was found to result in an increase of the solubilization yield of E. coli membranes, whereas in lipid-only model systems it decreased over the same pH range, based on optical density (OD) measurements. Similar opposite pH-dependent effects were observed in OD experiments comparing solubilization of native yeast membranes and yeast lipid-only membranes. We propose a model in which pH-dependent electrostatic interactions affect binding of the polymers to extramembraneous parts of membrane proteins, which in turn affects the availability of polymer for membrane solubilization. This model is supported by the observations that a similar pH-dependence as for SMA is observed for the anionic detergent SDS, but not for the nonionic detergent DDM and that the pH-dependence can be largely overcome by increasing the SMA concentration. The results are useful as guidelines to derive optimal conditions for solubilization of biological membranes by SMA.

Ceramides bind VDAC2 to trigger mitochondrial apoptosis.

Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity.

Membrane Solubilization by Styrene-Maleic Acid Copolymers: Delineating the Role of Polymer Length.

Styrene-maleic acid (SMA) copolymers have attracted interest in membrane research because they allow the solubilization and purification of membrane-spanning proteins from biological membranes in the form of native-like nanodisks. However, our understanding of the underlying SMA-lipid interactions is hampered by the fact that SMA preparations are very polydisperse. Here, we obtained fractions of the two most commonly used SMA preparations: SMA 2:1 and SMA 3:1 (both with specified Mw ∼10 kD), with different number-average molecular weight (Mn) and styrene content. The fractionation is based on the differential solubility of styrene-maleic anhydride (SMAnh) in hexane and acetone mixtures. SMAnh fractions were hydrolyzed to SMA and added to lipid self-assemblies. It was found that SMA fractions inserted in monolayers and solubilized vesicles to a different extent, with the highest efficiency being observed for low-Mn SMA polymers. Electron microscopy and dynamic light scattering size analyses confirmed the presence of nanodisks independent of the Mn of the SMA polymers forming the belt, and it was shown that the nanodisks all have approximately the same size. However, nanodisks bounded by high-Mn SMA polymers were more stable than those bounded by low-Mn polymers, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates of lipids between nanodisks. In conclusion, we here present a simple method to separate SMAnh molecules based on their Mn from commercial SMAnh blends, which allowed us to obtain insights into the importance of SMA length for polymer-lipid interactions.