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.

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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 M ∼10 kD), with different number-average molecular weight (M) 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-M SMA polymers. Electron microscopy and dynamic light scattering size analyses confirmed the presence of nanodisks independent of the M 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-M SMA polymers were more stable than those bounded by low-M 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 M from commercial SMAnh blends, which allowed us to obtain insights into the importance of SMA length for polymer-lipid interactions.

Mitochondria depend on phospholipids supplied by the endoplasmic reticulum, but how these lipids cross the outer membrane to enter mitochondria is unclear. We identified the voltage-dependent ion channel (VDAC) as a scramblase that facilitates lipid entry. Our analyses show that phospholipid transport across the outer membrane is impaired in yeast mitochondria lacking VDAC homologs. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 facilitate rapid scrambling, whereas VDAC1 monomers are less active. Molecular dynamics simulations of VDAC1 dimers support rapid scrambling at a dimer interface where polar residues induce large water defects and bilayer thinning. Thus, VDAC, a member of a superfamily of beta barrel proteins, moonlights as a new class of phospholipid scramblase operating by an unprecedented mechanism.

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 bilayers [1]. 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.