Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer ...membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB–TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.
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•Co-expression, mild extraction, and first EM insights of MFS-type EmrAB–TolC•EmrAB-TolC reveals a 33 nm long complex delineated by an Amphipol belt.•The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC.•There is a probable tip-to-tip interaction between EmrA and TolC.•TolC linked to the periplasmic adaptor EmrA forms an extended periplasmic canal.
Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these ...membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.
The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is ...unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.
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•Asters are ER-resident proteins that mediate nonvesicular sterol transport•The sterol-binding ASTER domain is structurally related to the START domain•Asters are dynamically recruited to PM-ER contacts in response to cholesterol•Aster-B is required for delivery of HDL cholesterol from SR-BI to the ER in adrenals
A nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals is mediated by sterol-binding ER-resident Aster proteins.
Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli. Their dynamic morphology is a result of regulated ...and balanced fusion and fission processes. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the 'neck' of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders.
Most antifungal peptides (AFPs), if not all, have membrane activity, while some also have alternative targets. Fungal membranes share many characteristics with mammalian membranes with only a few ...differences, such as differences in sphingolipids, phosphatidylinositol (PI) content and the main sterol is ergosterol. Fungal membranes are also more negative and a better target for cationic AFPs. Targeting just the fungal membrane lipids such as phosphatidylinositol and/or ergosterol by AFPs often translates into mammalian cell toxicity. Conversely, a specific AFP target in the fungal pathogen, such as glucosylceramide, mannosyldiinositol phosphorylceramide or a fungal protein target translates into high pathogen selectivity. However, a lower target concentration, absence or change in the specific fungal target can naturally lead to resistance, although such resistance in turn could result in reduced pathogen virulence. The question is then to be or not to be membrane active - what is the best choice for a successful AFP? In this review we deliberate on this question by focusing on the recent advances in our knowledge on how natural AFPs target fungi.
•Is membrane activity the best option for antifungal peptides (AFPs)?•Fungal membranes have unique lipids that are targeted by AFPs.•Targeting just fungal membrane lipids can translate into mammalian cell toxicity.•If an AFP only has one specific fungal target there is some potential for resistance.•Many AFPs target the fungal membrane, as well as other fungal components/systems.
Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains ...poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.
The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl ...inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.
A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) ...and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
Plant flavonoids are not only known as powerful antioxidants, but also as cell metabolism regulators. It has been postulated that they are able to control cell signal pathways by targeting receptors ...on the cell surface or by intercalating the lipid bilayer of membranes. Some flavonoids can increase lipid viscosity and decrease the cooperativity of hydrocarbon chain melting, while others can considerably decrease the lipid melting temperature, thus providing additional freedom for lipid diffusion. Here we discuss the ability of flavonoids to influence phase transition and lateral segregation of lipids, responsible for the formation of membrane compartments known as lipid rafts. The thermodynamic parameters of the bilayer determined by lipid packing characteristics and by lateral segregation of the bilayer are expected to depend on the location of flavonoid molecules in the bilayer. Flavonoid molecules preferably located in the hydrophobic region of the bilayer can initiate formation of raft-like domains (raft-making effect), while the molecules located in the polar interface region of the bilayer can fluidize membranes (raft-breaking effect), or initiate formation of interdigitated or micellar structures. Accordingly, we expect that in cellular membranes flavonoids can influence the appearance and development of rafts or raft-like membrane domains and thus influence the lateral diffusion of lipid molecules. Because rafts participate in cellular signal transduction, endocytosis and transmembrane translocation of different compounds, flavonoids may control cell metabolism by modulating the bilayer state.
Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates ...the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2 a -and Rab-A1 e labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.
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