β‐barrel membrane proteins in the mitochondrial outer membrane are crucial for mediating the metabolite exchange between the cytosol and the mitochondrial intermembrane space. In addition, the β‐barrel membrane protein subunit Tom40 of the translocase of the outer membrane (TOM) is essential for the import of the vast majority of mitochondrial proteins encoded in the nucleus. The sorting and assembly machinery (SAM) in the outer membrane is required for the membrane insertion of mitochondrial β‐barrel proteins. The core subunit Sam50, which has been conserved from bacteria to humans, is itself a β‐barrel protein. The β‐strands of β‐barrel precursor proteins are assembled at the Sam50 lateral gate forming a Sam50‐preprotein hybrid barrel. The assembled precursor β‐barrel is finally released into the outer mitochondrial membrane by displacement of the nascent β‐barrel, termed the β‐barrel switching mechanism. SAM forms supercomplexes with TOM and forms a mitochondrial outer‐to‐inner membrane contact site with the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. SAM shares subunits with the ER‐mitochondria encounter structure (ERMES), which forms a membrane contact site between the mitochondrial outer membrane and the endoplasmic reticulum. Therefore, β‐barrel membrane protein biogenesis is closely connected to general mitochondrial protein and lipid biogenesis and plays a central role in mitochondrial maintenance.

The majority of mitochondrial precursor proteins are imported through the Tom40 β-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for β-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction. These principles help to collect supernatant fractions with less cross-contaminations from the corresponding pellets. These approaches are scalable and adaptable for the fractionation of other cell types and are also useful for the characterization of other organelles.

The majority of mitochondrial precursor proteins are imported through the Tom40 β-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for β-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here we demonstrate that the a-helical outer membrane protein Mco6 forms a complex with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. Moreover, Mco6 also interacts with the subunit Mim1 of the mitochondrial import complex (MIM), which is itself required for the biogenesis of a-helical outer membrane proteins. MCO6 and MDM10 display a negative genetic interaction and a MCO6-MDM10 yeast double mutant contains reduced levels of TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and MIM-subunits associated with assembly defects of the TOM complex. Thus, this work reveals a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

Mitochondrial β-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic β-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last β-strand (β-signal) of the substrate-the 19-β-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first β-segment (β1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces β1 to promote its pairing with Tom40's last β-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial β-barrel folding.

The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria 1 , 2 . About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices 3 , 4 . TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. 5 – 7 ). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane.

The mitochondrial outer membrane contains so-called β-barrel proteins, which allow communication between the cytosol and the mitochondrial interior 1 – 3 . Insertion of β-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB) 4 – 6 . Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8–3.2 Å. The dimeric complex contains two copies of the β-barrel channel protein Sam50—Sam50a and Sam50b—with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the β-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed β-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a β-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of β-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for β-barrel assembly.

Nutrient transporters are prominent and ubiquitous components of the plasma membrane in all cell types. Their expression and regulation are tightly linked to the cells’ needs. Environmental factors such as nutrient starvation or osmotic stress prompt an acute remodeling of transporters and the plasma membrane to efficiently maintain homeostasis in cell metabolism. Lateral confinement of nutrient transporters through dynamic segregation within the plasma membrane has recently emerged as an important phenomenon that facilitates spatiotemporal control of nutrient uptake and metabolic regulation. Here, we review recent studies highlighting the mechanisms connecting the function of amino acid permeases with their endocytic turnover and lateral segregation within the plasma membrane. These findings indicate that actively controlled lateral compartmentalization of plasma membrane components constitutes an important level of regulation during acute cellular adaptations.

Lipid bilayers of different phospholipid compositions have been prepared, in the form of vesicles, or of supported lipid bilayers, and doped with Aurora (TM) at 0.1 mol%. Aurora (TM) consists of an Au-55 gold nanoparticle (about 1.4 nm in diameter) capped with triphenylphosphine ligands and a single diglyceride (distearoyl glycerol) ligand. Gold nanoparticles have been incorporated in the past inside liposomes, or grafted onto their surfaces, with diagnostic or therapeutic aims. Including the gold nanoparticles in a stable form within the lipid bilayers has serious technical difficulties. We have tested the hypothesis that, because of the diglyceride ligand, Aurora (TM) would allow the easy incorporation of gold nanoclusters into cell membranes or lipid bilayers. Our results show that Aurora (TM) readily incorporates into lipid bilayers, particularly when they are in the fluid phase, i.e. the state in which cell membranes exist. Calorimetric, fluorescence polarization or fluorescence confocal microscopy concur in showing that bilayer-embedded Aurora (TM) hardly changes the physical properties of the bilayers, nor does it perturb the phase equilibrium in lipid mixtures giving rise to lateral phase separation in the plane of the membrane. Atomic force microscopy shows, in fluid bilayers, well-resolved particles, 1.2-2.9 nm in height, that are interpreted as single Aurora (TM) conjugates. Cryo-transmission electron microscopy allows the clear observation of lipid bilayers with an enhanced contrast due to the Aurora (TM) gold nanoparticles; the single particles can be resolved at high magnification. Our studies support the applicability of Aurora (TM) as a membrane-friendly form of nano-gold particles for biological research or clinical applications.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

Sphingosine [(2 S,3 R,4 E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4 Z-sphingosine and palmitic acid ( cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer.

Biological membranes organize their proteins and lipids into nano- and microscale patterns. In the yeast plasma membrane (PM), constituents segregate into a large number of distinct domains. However, whether and how this intricate patchwork contributes to biological functions at the PM is still poorly understood. Here, we reveal an elaborate interplay between PM compartmentalization, physiological function, and endocytic turnover. Using the methionine permease Mup1 as model system, we demonstrate that this transporter segregates into PM clusters. Clustering requires sphingolipids, the tetraspanner protein Nce102, and signaling through TORC2. Importantly, we show that during substrate transport, a simple conformational change in Mup1 mediates rapid relocation into a unique disperse network at the PM Clustered Mup1 is protected from turnover, whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its own turnover. Our findings suggest that lateral compartmentalization provides an important regulatory link between function and turnover of PM proteins.

The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

Free volume voids in lipid bilayers can be measured by positron annihilation lifetime spectroscopy (PALS). This technique has been applied, together with differential scanning calorimetry and molecular dynamics (MD) simulations, to study the effects of cholesterol (Chol) and ceramide (Cer) on free volume voids in sphingomyelin (SM) or dipalmitoylphosphatidylcholine (DPPC) bilayers. Binary lipid samples with Chol were studied (DPPC:Chol 60:40, SM:Chol 60:40 mol ratio), and no phase transition was detected in the 20-60 degrees C range, in agreement with calorimetric data. Chol-driven liquid-ordered phase showed an intermediate free volume void size as compared to gel and fluid phases. For SM and SM:Cer (85:15 mol:mol) model membranes measured in the 20-60 degrees C range the gel-to-fluid phase transition could be observed with a related increase in free volume, which was more pronounced for the SM:Cer sample. MD simulations suggest a hitherto unsuspected lipid tilting in SM:Cer bilayers but not in pure SM. Ternary samples of DPPC:Cer:Chol (54:23:23) and SM:Cer:Chol (54:23:23) were measured, and a clear pattern of free volume increase was observed in the 20-60 degrees C because of the gel-to-fluid transition. Interestingly, MD simulations showed a tendency of Cer to change its distribution along the membrane to make room for Chol in ternary mixtures. The results suggest that the gel phase formed in these ternary mixtures is stabilized by Chol-Cer interactions.

Atomic force microscopy (AFM) has been applied to the characterization of palmitoylceramide (pCer) and cholesterol (Chol) incorporation into phospholipid-based supported planar bilayers (SPBs) at 22°C. Phospholipids were dipalmitoyl phosphatidylcholine (DPPC) or palmitoyl sphingomyelin (pSM). Membranes of different compositions were prepared by either the vesicle adsorption or the spin-coating procedures (the latter strictly for pCer-containing mixtures) and analyzed with a combination of AFM imaging (domain segregation, bilayer thickness and roughness) and force spectroscopy (mechanical resistance to indentation). The mixtures under study were pure phospholipids (pSM, DPPC), phospholipid:Chol (70:30), phospholipid:Chol:pCer (2:1:1) and phospholipid:pCer (90:10, 80:20 and 70:30). Binary phospholipid:pCer mixtures at increasing ceramide ratios gave rise to highly-resistant segregated domains with increasing extension but similar properties in terms of breakthrough forces, thicknesses and roughnesses. These ceramide-enriched domains are able to exclude a fluorescent lipid probe (DiIC18) due to their high intermolecular packing. Interestingly, these domains have been reported to disappear when model membranes become highly enriched in cholesterol in fluid membranes or in the absence of a fluid phase (our case). Indeed, the ternary mixtures (2:1:1) gave rise to a homogenous lamellar gel phase with significantly different properties when compared to all of the other mixtures under study: ternary mixtures showed a reduced thickness and an intermediate roughness and mechanical resistance when compared to phospholipid:Chol (70:30) and phospholipid:pCer. These differences were statistically significant. More importantly, at those relatively high pCer and Chol concentrations in ternary mixtures, no mutual displacement of these molecules was observed. The data become relevant in the context of sphingolipid signaling and membrane platform formation.

Protonation of the lumen-exposed residues of some photosynthetic complexes in the grana membranes occurs under conditions of high light intensity and triggers a major photoprotection mechanism known as energy dependent nonphotochemical quenching. We have studied the role of protonation in the structural reorganization and thermal stability of isolated grana membranes. The macroorganization of granal membrane fragments in protonated and partly deprotonated state has been mapped by means of atomic force microscopy. The protonation of the photosynthetic complexes has been found to induce large-scale structural remodeling of grana membranes-formation of extensive domains of the major light-harvesting complex of photosystem II and clustering of trimmed photosystem II supercomplexes, thinning of the membrane, and reduction of its size. These events are accompanied by pronounced thermal destabilization of the photosynthetic complexes, as evidenced by circular dichroism spectroscopy and differential scanning calorimetry. Our data reveal a detailed nanoscopic picture of the initial steps of nonphotochemical quenching.

Supported planar bilayers (SPBs) on mica substrates have been studied at 23 °C under atomic force microscopy (AFM)-based surface topography and force spectroscopy with two main objectives: (i) to characterize palmitoylceramide (pCer)-induced gel (Lβ) domains in binary mixtures with either its sphingolipid relative palmitoylsphingomyelin (pSM) or the glycerophospholipid dipalmitoylphosphorylcholine (DPPC) and (ii) to evaluate effects of incorporating cholesterol (Chol) into the previous mixtures in terms of Cer and Chol cooperation for the generation of lamellar gel (Lβ) phases of ternary composition. Binary phospholipid/pCer mixtures at XpCer < 0.33 promote the generation of laterally segregated micron-sized pCer-rich domains. Their analysis at different phospholipid/pCer ratios, by means of domain thickness, roughness, and mechanical resistance to tip piercing, reveals unvarying AFM-derived features over increasing pCer concentrations. These results suggest that the domains grow in size with increasing pCer concentrations while keeping a constant phospholipid/pCer stoichiometry. Moreover, the data show important differences between pCer interactions with pSM or DPPC. Gel domains generated in pSM/pCer bilayers are thinner than the pSM-rich surrounding phase, while the opposite is observed in DPPC/pCer mixtures. Furthermore, a higher breakthrough force is observed for pSM/pCer as compared to DPPC/pCer domains, which can be associated with the preferential pCer interaction with its sphingolipid relative pSM. Cholesterol incorporation into both binary mixtures at a high Chol and pCer ratio abolishes any phospholipid/pCer binary domains. Bilayers with properties different from any of the pure or binary samples are observed instead. The data support no displacement of Chol by pCer or vice versa under these conditions, but rather a preferential interaction between the two hydrophobic lipids.

Three ceramide analogues have been synthesized, with sphingosine-like chains containing five conjugated double bonds. Pentaene I has an N-palmitoyl acyl chain, while the other two pentaenes contain also a doxyl radical, respectively, at C5 (Penta5dox) and at C16 (Penta16dox) positions of the N-acyl chain. Pentaene I maximum excitation and emission wavelengths in a phospholipid bilayer are 353 and 478 nm, respectively. Pentaene I does not segregate from the other lipids in the way natural ceramide does, but rather mixes with them in a selective way according to the lipid phases involved. Fluorescence confocal microscopy studies show that when lipid domains in different physical states coexist, Pentaene I emission is higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Electron paramagnetic resonance of the pentaene doxyl probes confirms that these molecules are sensitive to the physical state of the bilayer. Calorimetric and fluorescence quenching experiments suggest that the lipids under study orient themselves in lipid bilayers with their polar moieties located at the lipid water interface. The doxyl radical in the N-acyl chain quenches the fluorescence of the pentaene group when in close proximity. Because of this property, Penta16dox can detect gel fluid transitions in phospholipids. The availability of probes for lipids in the gel phase is important in view of novel evidence for the existence of gel microdomains in cell membranes.