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BBA - Biomembranes (v.1808, #3)

Editorial Board (pp. i).

Sodium–calcium exchanger and lipid rafts in pig coronary artery smooth muscle by Iwona Kuszczak; Sue E. Samson; Jyoti Pande; Dana Q. Shen; Ashok K. Grover (pp. 589-596).

Pig coronary artery smooth muscle expresses, among many other proteins, Na⁺–Ca²⁺-exchanger NCX1 and sarcoplasmic reticulum Ca²⁺ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca²⁺ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca²⁺ for refilling the sarcoplasmic reticulum.► Lipid rafts in the plasma membrane of coronary artery smooth muscle are of two types. ► The first type of lipid rafts are rich in caveolin and flotillin. ► The second type is rich in cholesterol, GM gangliosides and prion proteins. ► NCX1 and SERCA2 are co-localized in the first. ► NCX1–SERCA2 co-localization may aid in cell signaling.

Keywords: Abbreviations; Ca; ²⁺; i; cytosolic Ca; ²⁺; EGTA; ethyleneglycol bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; NCX; Na; ⁺; –Ca; ²⁺; -exchanger; PBS; phosphate buffered saline; PM; plasma membrane; PMCA; PM Ca; ²⁺; pump; SR; sarcoplasmic reticulum; SERCA; SR Ca; ²⁺; pumpNCX1; SERCA2; Caveolin; Fluorescence microscopy; Lipid raft

Sodium–calcium exchanger and lipid rafts in pig coronary artery smooth muscle by Iwona Kuszczak; Sue E. Samson; Jyoti Pande; Dana Q. Shen; Ashok K. Grover (pp. 589-596).

Ouabain-induced modifications of prostate cancer cell lipidome investigated with mass spectrometry and FTIR spectroscopy by Régis Gasper; Guy Vandenbussche; Erik Goormaghtigh (pp. 597-605).

Fourier transform infrared (FTIR) spectroscopy was used to investigate modifications of prostate cancer PC-3 cell lipidome after exposure to sub-lethal concentrations of ouabain. FTIR spectroscopy offered an overview of the lipid classes present in the whole sample. The method is simple, label free and some features can be detected on entire cells. We compared the achievements of FTIR spectroscopy with data obtained by mass spectrometry (MS) on the same samples. It appears that FTIR spectroscopy could identify content variations in some lipid classes, e.g., these containing choline head groups such as phosphatidylcholine and sphingomyelin. MS analysis could confirm this result as indicated by principal component analysis and 2D heterocorrelation maps. FTIR spectra were also able to report changes in ester/choline/phosphate ratios characterizing lipid changes induced by ouabain. Furthermore, quantization of major lipid classes (PC, PE, PG, SM) could be obtained by curve fitting of the FTIR spectra. Yet, FTIR failed to resolve lipid classes for which the polar heads do not display specific IR features such as phosphatidylglycerol and cardiolipin.► Lipid composition. ► PC3 prostate cancer cells. ► Mass spectrometry.

Keywords: Abbreviations; FTIR; Fourier transform infrared; MS; mass spectrometry; PCA; principal component analysis; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PS; phosphatidylserine; SM; sphingomyelin; PI3K; phosphatidylinositol-3 kinaseIR spectroscopy; Mass spectrometry; Ouabain; Cancer; Drug; Lipid; Hetero-spectral correlation

Ouabain-induced modifications of prostate cancer cell lipidome investigated with mass spectrometry and FTIR spectroscopy by Régis Gasper; Guy Vandenbussche; Erik Goormaghtigh (pp. 597-605).

Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks by Chung-Ping Leon Wan; Michael H. Chiu; Xinping Wu; Sean K. Lee; Elmar J. Prenner; Paul M.M. Weers (pp. 606-613).

Apolipoprotein mediated formation of nanodisks was studied in detail using apolipophorin III (apoLp-III), thereby providing insight in apolipoprotein–lipid binding interactions. The spontaneous solubilization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles occured only in a very narrow temperature range at the gel–liquid–crystalline phase transition temperature, exhibiting a net exothermic interaction based on isothermal titration calorimetry analysis. The resulting nanodisks were protected from proteolysis by trypsin, endoproteinase Glu-C, chymotrypsin and elastase. DMPC solubilization and the simultaneous formation of nanodisks were promoted by increasing the vesicle diameter, protein to lipid ratio and concentration. Inclusion of cholesterol in DMPC dramatically enhanced the rate of nanodisk formation, presumably by stabilization of lattice defects which form the main insertion sites for apolipoprotein α-helices. The presence of fully saturated acyl chains with a length of 13 or 14 carbons in phosphatidylcholine allowed the spontaneous vesicle solubilization upon apolipoprotein addition. Nanodisks with C13:0-phosphatidylcholine were significantly smaller with a diameter of 11.7±3.1nm compared to 18.5±5.6nm for DMPC nanodisks determined by transmission electron microscopy. Nanodisk formation was not observed when the phosphatidylcholine vesicles contained acyl chains of 15 or 16 carbons. However, using very high concentrations of lipid and protein (>10mg/ml), 1,2,-dipalmitoyl-sn-glycero-3-phosphocholine nanodisks could be produced spontaneously although the efficiency remained low.► Apolipophorin III spontaneously solubilizes phosphatidylcholine vesicles. ► ITC shows exothermic character of formation of DMPC nanodisks. ► Nanodisks are well protected from proteases. ► Cholesterol and sphingomyelin stimulate nanodisk formation. ► Nanodisk formation is efficient process for PC with acylchains of 14C or shorter.

Keywords: Abbreviations; apoA-I; apolipoprotein A-I; apoLp-III; apolipophorin III; DG; 1,2-dimyristoyl-sn-glycerol; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; HDL; high density lipoprotein; ITC; isothermal titration calorimetry; LUV; large unilamellar vesicle; MLV; multilamellar vesicle; PBS; phosphate buffered saline; PC; phosphatidylcholine; SM; sphingomyelin; TEM; transmission electron microscopyApolipoprotein; Apolipophorin III; DMPC; Nanodisk; Phosphatidylcholine

A ToF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems by Eleonora Keating; Alan J. Waring; Frans J. Walther; Fred Possmayer; Ruud A.W. Veldhuizen; Nils O. Petersen (pp. 614-621).

Pulmonary surfactant is a complex lipid–protein mixture whose main function is to reduce the surface tension at the air–liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir–Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid–lipid and lipid–protein interactions. This may further our understanding of pulmonary surfactant function.► ToF-SIMS is suitable in studies of lateral organization of surfactant films. ► Saturated phospholipids (PLs) and cholesterol are found in LC domains. ► Unsaturated PLs and miniB are found in LE phase. ► Distribution of PLs and peptide not affected by cholesterol addition. ► ToF-SIMS identifies differences in organization of normal and dysfunctional PS.

Keywords: Pulmonary surfactant; Time-of-flight secondary mass spectrometry; Atomic force microscopy; Lipids proteins lateral organization; Cholesterol; Surfactant function

The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants by John T.J. Cheng; John D. Hale; Melissa Elliott; Robert E.W. Hancock; Suzana K. Straus (pp. 622-633).

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-(1′- rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and³¹P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.► The structure of aurein 2.2 and variants in different membranes is investigated. ► The membrane interactions of these peptides are also determined. ► The model membranes used are cardiolipin/PG and PE/PG. ► Results are compared to previous findings in PC/PG. ► Antimicrobial activity is correlated to the biophysical findings.

Keywords: Aurein 2.2; Aurein 2.3; Oriented circular dichroism; Solid state NMR; α-Helical structure; Membrane interaction; Membrane composition; Antimicrobial activity

Widefield microscopy for live imaging of lipid domains and membrane dynamics by Guy Wheeler; Kevin M. Tyler (pp. 634-641).

Within the lateral organisation of plasma membranes of polarized cell types there exist heterogenous microdomains of distinct lipid composition, the small size of which (10–200nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes.► Adaptation of wide-field microscope setup for visualising liquid-ordered domains. ► Real-time live cell imaging of plasma membrane heterogeneity and dynamics. ► Multi-channel simultaneous acquisition of biological markers with GP images.

Keywords: Abbreviations; GP; generalized polarization; l; _o; liquid-ordered; MDCKII; Madin–Darby Canine Kidney IILaurdan; Lipid raft; Widefield fluorescent imaging

Widefield microscopy for live imaging of lipid domains and membrane dynamics by Guy Wheeler; Kevin M. Tyler (pp. 634-641).

Keywords: Abbreviations; GP; generalized polarization; l; _o; liquid-ordered; MDCKII; Madin–Darby Canine Kidney IILaurdan; Lipid raft; Widefield fluorescent imaging

Membrane hydraulic permeability changes during cooling of mammalian cells by Maryam Akhoondi; Harriëtte Oldenhof; Christoph Stoll; Harald Sieme; Willem F. Wolkers (pp. 642-648).

In order to predict optimal cooling rates for cryopreservation of cells, the cell-specific membrane hydraulic permeability and corresponding activation energy for water transport need to be experimentally determined. These parameters should preferably be determined at subzero temperatures in the presence of ice. There is, however, a lack of methods to study membrane properties of cells in the presence of ice. We have used Fourier transform infrared spectroscopy to study freezing-induced membrane dehydration of mouse embryonic fibroblast (3T3) cells and derived the subzero membrane hydraulic permeability and the activation energy for water transport from these data. Coulter counter measurements were used to determine the suprazero membrane hydraulic permeability parameters from cellular volume changes of cells exposed to osmotic stress. The activation energy for water transport in the ice phase is about three fold greater compared to that at suprazero temperatures. The membrane hydraulic permeability at 0°C that was extrapolated from suprazero measurements is about five fold greater compared to that extrapolated from subzero measurements. This difference is likely due to a freezing-induced dehydration of the bound water around the phospholipid head groups. Using Fourier transform infrared spectroscopy, two distinct water transport processes, that of free and membrane bound water, can be identified during freezing with distinct activation energies. Dimethylsulfoxide, a widely used cryoprotective agent, did not prevent freezing-induced membrane dehydration but decreased the activation energy for water transport.► The activation energy for water transport across membranes increases at subzero temperatures. ► Two different water transport processes can be identified during freezing of cells. ► DMSO does not prevent membrane dehydration during freezing. ► DMSO decreases the activation energy for water transport during freezing.

Keywords: Abbreviations; E; _Lp; activation energy of the cell membrane permeability to water; FTIR; Fourier transform infrared spectroscopy; Lp; membrane hydraulic permeability; Lpg; membrane hydraulic permeability at 0; °C; Tn; ice nucleation temperature; V; _o; isosmotic cell volume; V; _b; osmotically inactive cell volumeFourier transform infrared spectroscopy; Lipid phase behavior; Suprazero and subzero membrane hydraulic permeability; Cryobiology

Membrane hydraulic permeability changes during cooling of mammalian cells by Maryam Akhoondi; Harriëtte Oldenhof; Christoph Stoll; Harald Sieme; Willem F. Wolkers (pp. 642-648).

Interaction of cationic bilayer fragments with a model oligonucleotide by Julio H.K. Rozenfeld; Tiago R. Oliveira; M. Teresa Lamy; Ana M. Carmona-Ribeiro (pp. 649-655).

The interaction between cationic bilayer fragments and a model oligonucleotide was investigated by differential scanning calorimetry, turbidimetry, determination of excimer to monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidyl-choline in bilayer fragment dispersions and dynamic light scattering for sizing and zeta-potential analysis. Salt (Na₂HPO₄), mononucleotide (2′-deoxyadenosine-5′-monophosphate) or poly (dA) oligonucleotide (3′-AAA AAA AAA A-5′) affected structure and stability of dioctadecyldimethylammonium bromide bilayer fragments. Oligonucleotide and salt increased bilayer packing due to bilayer fragment fusion. Mononucleotide did not reduce colloid stability or did not cause bilayer fragment fusion. Charge neutralization of bilayer fragments by poly (dA) at 1:10 poly (dA):dioctadecyldimethylammonium bromide molar ratio caused extensive aggregation, maximal size and zero of zeta-potential for the assemblies. Above charge neutralization, assemblies recovered colloid stability due to charge overcompensation. For bilayer fragments/poly (dA), the nonmonotonic behavior of colloid stability as a function of poly (dA) concentration was unique for the oligonucleotide and was not observed for Na₂HPO₄ or 2′-deoxyadenosine-5′-monophosphate. For the first time, such interactions between cationic bilayer fragments and mono- or oligonucleotide were described in the literature. Bilayer fragments/oligonucleotide assemblies may find interesting applications in drug delivery.► Oligonucleotide induced fusion of cationic bilayer fragments. ► Mononucleotide did not induce fusion of bilayer fragments. ► Charge neutralization of fragments by oligonucleotide caused extensive aggregation. ► Increasing oligonucleotide concentration restabilized fragments/oligonucleotide assemblies. ► Oligonucleotide adsorption increased bilayer packing due to BF fusion.

Keywords: Dioctadecyldimethylammonium bromide; 3′-AAA AAA AAA A-5′ oligonucleotide; 2′-deoxyadenosine-5′-monophosphate; Differential scanning calorimetry; Dynamic light scattering; Fusion of bilayer fragments

Interaction of cationic bilayer fragments with a model oligonucleotide by Julio H.K. Rozenfeld; Tiago R. Oliveira; M. Teresa Lamy; Ana M. Carmona-Ribeiro (pp. 649-655).

Biomimetic liposomes and planar supported bilayers for the assessment of glycodendrimeric porphyrins interaction with an immobilized lectin by A. Makky; J.P. Michel; Ph. Maillard; V. Rosilio (pp. 656-666).

Photodynamic therapy is a potentially efficient treatment for various solid tumours, among which retinoblastoma. Its efficacy depends on the preferential accumulation of photosensitizers in the malignant tissues and their accessibility to light. The specificity of drugs for retinoblastoma cells can be improved by targeting a mannose receptor overexpressed at their surface. With the aim of assessing the recognition of newly synthesized glycodendrimeric porphyrins by such receptors, we have built and characterized an original synthetic biomimetic membrane having similar lipidic composition to that of the retinal cell membranes and bearing Concanavalin A, as a model of the mannose receptor. The interaction of the porphyrin derivatives with liposomes and supported planar bilayers has been studied by dynamic light scattering and quartz crystal microbalance with dissipation monitoring (QCM-D). Only mannosylated porphyrins interacted significantly with the membrane model. The methodology used proved to be efficient for the selection of potentially active compounds.Display Omitted► Design of original biomimetic membrane models of retinoblastoma cell membrane. ► In situ grafting of mannose-specific lectin mimicking cells native receptors. ► Study of dendrimeric porphyrins interactions with the model membrane. ► Predominance of the specific interaction over the non-specific one. ► After binding, spacer length affects the viscoelastic properties of a system.

Keywords: Abbreviations; PDT; photodynamic therapy; α-MMP; methyl; α; -; d; -mannopyranoside; Con A; Concanavalin A; QCM-D; Quartz crystal microbalance with dissipation monitoring; DLS; dynamic light scattering; SPB; supported planar bilayerPDT; Retinoblastoma; Glycoconjugated porphyrins; Supported planar bilayer; Liposome; Lectin–sugar interaction

Insights into the biophysical properties of GABA_A ion channels: Modulation of ion permeation by drugs and protein interactions by M. Louise Tierney (pp. 667-673).

The fundamental properties of ion channels assure their selectivity for a particular ion, its rapid permeation through a central pore and that such electrical activity is modulated by factors that control the opening and closing (gating) of the channel. All cell types possess ion channels and their regulated flux of ions across the membrane play critical roles in all steps of life. An ion channel does not act alone to control cell excitability but rather forms part of larger protein complexes. The identification of protein interaction partners of ion channels and their influence on both the fundamental biophysical properties of the channel and its expression in the membrane are revealing the many ways in which electrical activity may be regulated. Highlighted here is the novel use of the patch clamp method to dissect out the influence of protein interactions on the activity of individual GABA_A receptors. The studies demonstrate that ion conduction is a dynamic property of a channel and that protein interactions in a cytoplasmic domain underlie the channel's ability to alter ion permeation. A structural model describing a reorganisation of the conserved cytoplasmic gondola domain and the influence of drugs on this process are presented.► Ion permeation through GABA_A receptors is variable. ► Drugs and protein interactions modulate ion permeation through clustered channels. ► These data are reviewed and a model is presented. ► Permeation through the membrane pore is linked to intracellular protein interactions. ► Cellular signals that affect GABA_A receptor interactions will affect neuronal function.

Keywords: Abbreviations; GABA; _A; receptor; γ-amino butyric acid type A receptor; MA helix; amphipathic helixGABA; _A; receptor; Conductance; Diazepam; Electrophysiology; MA helix; Peptide

Insights into the biophysical properties of GABA_A ion channels: Modulation of ion permeation by drugs and protein interactions by M. Louise Tierney (pp. 667-673).

Keywords: Abbreviations; GABA; _A; receptor; γ-amino butyric acid type A receptor; MA helix; amphipathic helixGABA; _A; receptor; Conductance; Diazepam; Electrophysiology; MA helix; Peptide

Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments by Eugenia Polverini; Eoin P. Coll; D. Peter Tieleman; George Harauz (pp. 674-683).

The 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.Display Omitted► MD simulations of conserved segment of 18.5 kDa MBP in different environments. ► Aqueous: phosphorylation stabilizes hydrophobic cluster, with formation of β-structures. ► DMPC bilayer: amphipathic α-helix stable & presents an SH3-ligand to cytoplasm. ► Phosphorylation alters accessibility of SH3-ligand & can stabilize a β-bend. ► Bend can explain how a single protein can bring two bilayers close together in myelin.

Keywords: Abbreviations; CD; circular dichroism; DMPC; dimyristoylphosphatidylcholine; EPR; electron paramagnetic resonance; MBP; myelin basic protein; MD; molecular dynamics; NMR; nuclear magnetic resonance; PPII; poly-proline type II conformation; PhT; phosphorylated threonine; RMSD; root mean square deviationMolecular dynamics; Myelin basic protein; Phosphorylation; PPII structure; Intrinsically disordered protein

Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP₂ by Alessio Ausili; Corbalan-Garcia Senena Corbalán-García; Gomez-Fernandez Juan C. Gómez-Fernández; Derek Marsh (pp. 684-695).

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP₂. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A₂. PIP₂ did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A₂ did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP₂, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.► The tilt of a β-sandwich protein docked on a lipid membrane was calculated. ► ATR-IR was used to calculate the tilt of the C2 domain of PKCα. ► The tilt of the β-sandwich was α=35° when bound to POPC/POPS membrane. ► When PIP2 is incorporated into the membrane the tilt angle increases to α=44°.

Keywords: Abbreviations; PKC; protein kinase C; cPLA; ₂; cytosolic phospholipase A; ₂; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoserine; PI; phosphatidylinositol; PIP; ₂; phosphatidylinositol 4,5-bisphosphate; DAG; diacylglycerol; PDB; protein data bank; PtdIns(3,4,5)P; ₃; phosphatidylinositol 3,4,5-trisphosphate; POPA; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphate; ATR; attenuated total internal reflection; IR; infrared; EPR; electron paramagnetic resonanceATR-IR; C2 domain; PIP; ₂; PKC

Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN by M. Victoria Picardi; Antonio Cruz; Guillermo Orellana; Perez-Gil Jesús Pérez-Gil (pp. 696-705).

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air–liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37°C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25°C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37°C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37°C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37°C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.Display Omitted► Packing in surfactant films are monitored by the fluorescence of the probe LAURDAN. ► Compression-driven packing of surfactant at 25 °C is similar to that of pure DPPC films. ► At 37 °C, maximal packing required the presence of cholesterol in surfactant films. ► Compressed films are more hydrated than bilayers of same composition, specially at 37 °C.

Keywords: Surface tension; Monolayer; Lung surfactant; Cholesterol; Air–liquid interface

High water solubility and fold in amphipols of proteins with large hydrophobic regions: Oleosins and caleosin from seed lipid bodies by Yann Gohon; Jean-David Vindigni; Agnès Pallier; Frank Wien; Hervé Celia; Alexandre Giuliani; Christophe Tribet; Thierry Chardot; Pierre Briozzo (pp. 706-716).

Seed lipid bodies constitute natural emulsions stabilized by specialized integral membrane proteins, among which the most abundant are oleosins, followed by the calcium binding caleosin. These proteins exhibit a triblock structure, with a highly hydrophobic central region comprising up to 71 residues. Little is known on their three-dimensional structure. Here we report the solubilization of caleosin and of two oleosins in aqueous solution, using various detergents or original amphiphilic polymers, amphipols. All three proteins, insoluble in water buffers, were maintained soluble either by anionic detergents or amphipols. Neutral detergents were ineffective. In complex with amphipols the oleosins and caleosin contain more beta and less alpha secondary structures than in the SDS detergent, as evaluated by synchrotron radiation circular dichroism. These are the first reported structural results on lipid bodies proteins maintained in solution with amphipols, a promising alternative to notoriously denaturing detergents.► Three integral proteins from lipid bodies efficiently solubilized in water. ► First structural results on lipid bodies proteins in solution with amphipols. ► Oleosins and caleosin contain more beta structures in amphipols than in SDS. ► Model for the secondary structure of the long hydrophobic domain of oleosins.

Keywords: Lipid body; Oleosin; Caleosin; Amphiphilic polymer; Secondary structure

Non-reducing trisaccharide fatty acid monoesters: Novel detergents in membrane biochemistry by Perez-Victoria Ignacio Pérez-Victoria; Perez-Victoria Francisco J. Pérez-Victoria; Roldan-Vargas Sándalo Roldán-Vargas; Garcia-Hernandez Raquel García-Hernández; Luis Carvalho; Santiago Castanys; Francisco Gamarro; Juan C. Morales; Perez-Victoria José M. Pérez-Victoria (pp. 717-726).

Three families of non-reducing trisaccharide fatty acid monoesters bearing C₁₀ to C₁₈ acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospolipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β -dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.► The new surfactants, regioisomerically pure, cover CMCs from μM to mM. ► They solubilize membrane proteins independently of their lipidic environment. ► They keep the activity of solubilized proteins better than classical detergents. ► They are fully compatible with chromatographic protein purification methods. ► Large unilamellar (proteo)liposomes are produced by their enzymatic hydrolysis.

Keywords: Abbreviations; ABC; ATP-binding cassette; MDR; multidrug resistance; Pgp; P-glycoprotein; Ni-NTA; nickel nitriloacetic acid; HLB; hydrophilic–lipophilic balance; CMC; critical micelle concentration; DDM; β; -; dodecylmaltoside; OG; β; -; octylglucoside; SUL; sucrose monolaurate; R6D; 6-; O; -decanoylraffinose; R6L; 6-; O; -lauroylraffinose; R6M; 6-; O; -myristoylraffinose; R6P; 6-; O; -palmytoylraffinose; R6S; 6-; O; -stearoylraffinose; R1D; 1″-; O; -decanoylraffinose; R1L; 1″-; O; -lauroylraffinose; R1M; 1″-; O; -myristoylraffinose; R1P; 1″-; O; -palmytoylraffinose; R1S; 1″-; O; -stearoylraffinose; MD; decanoylmelezitose; ML; lauroylmelezitose; MM; myristoylmelezitose; MP; palmitoylmelezitose; MS; stearoylmelezitoseCarbohydrate fatty acid monoesters; Detergents; Membrane protein purification; (Proteo)liposomes; ABC transporters

Membrane bilayer properties of sphingomyelins with amide-linked 2- or 3-hydroxylated fatty acids by Oscar Ekholm; Shishir Jaikishan; Lonnfors Max Lönnfors; Thomas K.M. Nyholm; J. Peter Slotte (pp. 727-732).

The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(d)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5–10°C) gel–liquid transition temperatures ( T_m) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(d)-palmitoyl SM, however, had lower T_m (5°C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner.► Acyl chain-hydroxylated sphingomyelins (SM) exist in specialized tissues and cells, and their biophysical and sterol-interacting properties were examined. ► It was clearly observed that 2-hydroxylation stabilized SM interactions, whereas 3-hydroxylation destabilized, irrespective of chain length. ► Sterol interaction with both 2- and 3-hydroxylated SM analogs was attenuated compared to nonhydroxylated SM. ► We conclude that hydroxylation in the SM interfacial region markedly affects intermolecular interactions and SM/sterol association.

Keywords: Abbreviations; 22:0-SM; N; -behenoyl-; d; -; erythro; -sphingomyelin; 2OH-22:0-SM; N; -2OH(; d; )-behenoyl-; d; -; erythro; -sphingomyelin; 2OH-PSM; N; -2OH(; d; )-palmitoyl-; d; -; erythro; -sphingomyelin; 3OH-PSM; N; -3OH(; d; )-palmitoyl-; d; -; erythro; -sphingomyelin; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholesta-5,7(11)-trien-3-beta-ol; DPH; 1,6-diphenyl-1,3,5-hexatriene; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; N; -palmitoyl-; d; -; erythro; -sphingomyelinHydroxylated sphingomyelin; Cholesterol; Bilayer membrane; Lateral interaction; Ordered domains; Sterol affinity

Over-expression of functional Saccharomyces cerevisiae GUP1, induces proliferation of intracellular membranes containing ER and Golgi resident proteins by Gianluca Bleve; Gian Pietro Di Sansebastiano; Francesco Grieco (pp. 733-744).

High-level expression of the GUP1 gene in Saccharomyces cerevisiae resulted in the formation of proliferated structures, which hosted endoplasmic reticulum (ER), Golgi and itinerant proteins. The GUP1 over-expression enhanced ER biogenesis, as shown by the coordinated increased transcription rate of genes involved in both ER and Golgi metabolism and in phospholipids biosynthesis. The formation of Gup1-induced proliferation revealed that it depended on an intact unfolded protein response, because their assembly was reported to be lethal to yeast strains unable to initiate the UPR (Unfolded Protein Response) pathway. GUP1 over-expression affected global ER and Golgi structure and resulted in the biogenesis of novel membrane arrays with Golgi and ER hybrid composition. In fact, a number of ER and Golgi resident proteins together with itinerant proteins that normally cycle between ER and Golgi, were localized in the proliferated stacked membranes. The described assembling of novel membrane structures was affected by the functionality of the Gup1 O-acyltransferase domain, which regulates the Gup1 protein role as remodelase in the glycosylphosphatidylinositol (GPI) anchored proteins biosynthesis. To our knowledge, we presented the first evidence of sub cellular modifications in response over-expression of a GPI-anchor remodelase in S. cerevisiae.► High-level expression of GUP1 gene in S. cerevisiae resulted in the formation of proliferated structures. ► The Gup1 protein-induced proliferations showed that they are a hybrid compartment, including resident ER, Golgi and itinerant proteins. ► The Gup1p MBOAT activity was needed for the membranaceous arrangements formation.

Keywords: GUP1; Yeast; Endoplasmic reticulum; Membrane biogenesis; Yeast membrane proteins; Unfolded protein response

The predominant role of IP₃ type 1 receptors in activation of store-operated Ca²⁺ entry in liver cells by L. Jones; L. Ma; J. Castro; T. Litjens; G.J. Barritt; G.Y. Rychkov (pp. 745-751).

Physiologically, hormone induced release of Ca²⁺ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP₃) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP₃ receptors act as channels, releasing Ca²⁺ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca²⁺ stores leads to activation of store-operated Ca²⁺ channels on the plasma membrane which replenishes lost Ca²⁺ and sustain Ca²⁺ signalling. There are three isoforms of IP₃ receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca²⁺ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca²⁺ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca²⁺ channels, and Ca²⁺ release activated Ca²⁺ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP₃ receptor but not the others. Here we show that H4IIE liver cells express all three types of IP₃ receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I_CRAC), while type 1 and type 2, but not type 3, participate in observed Ca²⁺ release in response to receptor stimulation . Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca²⁺ store linked to the activation of I_CRAC predominantly expresses type 1 IP₃ receptors.►H4IIE liver cells express all three types of IP₃ receptor. ►IP₃R type 1 and to a lesser extent type 3, but not type 2, participate in activation of I_CRAC. ►IP₃R type 1 and type 2, but not type 3, participate in observed Ca²⁺ release in response to receptor stimulation. ►In H4IIE rat liver cells the sub-compartment of intracellular Ca²⁺ store linked to activation of I_CRAC predominantly expresses type 1 IP₃ receptors.

Keywords: Abbreviations; IP; ₃; inositol 1,4,5-trisphosphate; IP; ₃; Rs; inositol 1,4,5-trisphosphate receptors; CRAC; Ca; ²⁺; release activated Ca; ²⁺; SOCE; store-operated Ca; ²⁺; entry; Rn; rattus norvegicusCRAC channels; IP; ₃; receptor; Ca; ²⁺; stores

The predominant role of IP₃ type 1 receptors in activation of store-operated Ca²⁺ entry in liver cells by L. Jones; L. Ma; J. Castro; T. Litjens; G.J. Barritt; G.Y. Rychkov (pp. 745-751).

Plant pentacyclic triterpenic acids as modulators of lipid membrane physical properties by Jesús Prades; Vogler Oliver Vögler; Regina Alemany; Manuel Gomez-Florit; Sérgio S. Funari; Ruiz-Gutierrez Valentina Ruiz-Gutiérrez; Barcelo Francisca Barceló (pp. 752-760).

Free triterpenic acids (TTPs) present in plants are bioactive compounds exhibiting multiple nutriceutical activities. The underlying molecular mechanisms have only been examined in part and mainly focused on anti-inflammatory properties, cancer and cardiovascular diseases, in all of which TTPs frequently affect membrane-related proteins. Based on the structural characteristics of TTPs, we assume that their effect on biophysical properties of cell membranes could play a role for their biological activity. In this context, our study is focused on the compounds, oleanolic (3β-hydroxy-12-oleanen-28-oic acid, OLA), maslinic (2α,3β-dihydroxy-12-oleanen-28-oic acid, MSL) and ursolic ((3β)-3-hydroxyurs-12-en-28-oic acid, URL) as the most important TTPs present in orujo olive oil. X-ray diffraction, differential scanning calorimetry,³¹P nuclear magnetic resonance and Laurdan fluorescence data provide experimental evidence that OLA, MSL and URL altered the structural properties of 1,2-dipalmitoyl -sn-glycero-3-phosphatidylcholine (DPPC) and DPPC-Cholesterol (Cho) rich membranes, being located into the polar-hydrophobic interphase. Specifically, in DPPC membranes, TTPs altered the structural order of the L_β′, phase without destabilizing the lipid bilayer. The existence of a nonbilayer isotropic phase in coexistence with the liquid crystalline L_α phase, as observed in DPPC:URL samples, indicated the presence of lipid structures with high curvature (probably inverted micelles). In DPPC:Cho membranes, TTPs affected the membrane phase properties increasing the Laurdan GP values above 40°C. MSL and URL induced segregation of Cho within the bilayer, in contrast to OLA, that reduced the structural organization of the membrane. These results strengthen the relevance of TTP interactions with cell membranes as a molecular mechanism underlying their broad spectrum of biological effects.► Plant triterpenic acids (TTPs) alter the structural properties of PC membranes. ► Phase properties of PC-cholesterol membranes are sensitive to TTP-interaction. ► Differences in TTP-lipid interactions are related to their chemical structure.

Keywords: Abbreviations; TTP; free triterpenic acid; PL; phospholipids; PC; phosphatidylcholine; DPPC; 1,2-dipalmitoyl; -sn-; glycero-3-phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; DEPE; 1,2-dielaidoyl; -sn; -glycero; -; 3-phosphatidylethanolamine; Cho; cholesterol; OLA; oleanolic acid; MSL; maslinic acid; URL; ursolic acid; Laurdan; 6-dodecanoyl-2-(dimethylamino)naphthalene; DPH; 1,6-diphenyl-1,3,5-hexatriene; MLV; multilamellar lipid vesicles; SUV; small unilamellar vesicles; DSC; differential scanning calorimetry; L; _β′; gel lamellar phase; P; _β′; rippled phase; L; _α; liquid-crystalline lamellar phase; H; _II; inverted hexagonal phase; d; bilayer repeat period; T; _p; lamellar gel-to-rippled phase transition temperature; T; _m; gel-to-liquid crystalline phase transition temperature; T; _H; lamellar-to-inverted hexagonal phase transition temperature; ΔH; enthalpy changePlant triterpenic acid; Triterpene–membrane interaction; Membrane structure

Myelin basic protein binds microtubules to a membrane surface and to actin filaments in vitro: Effect of phosphorylation and deimination by Joan M. Boggs; Godha Rangaraj; Yew-Meng Heng; Yuanfang Liu; George Harauz (pp. 761-773).

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. It assembles actin filaments and microtubules, can bind actin filaments and SH3-domains to a membrane surface, and may be able to tether them to the oligodendrocyte membrane and participate in signal transduction in oligodendrocytes/myelin. In the present study, we have shown that the 18.5kDa MBP isoform can also bind microtubules to lipid vesicles in vitro. Phosphorylation of MBP at Thr94 and Thr97 (bovine sequence) by MAPK, and deimination of MBP (using a pseudo-deiminated recombinant form), had little detectable effect on its ability to polymerize and bundle microtubules, in contrast to the effect of these modifications on MBP-mediated assembly of actin. However, these modifications dramatically decreased the ability of MBP to tether microtubules to lipid vesicles. MBP and its phosphorylated and pseudo-deiminated variants were also able to bind microtubules to actin filaments. These results suggest that MBP may be able to tether microtubules to the cytoplasmic surface of the oligodendrocyte membrane, and that this binding can be regulated by post-translational modifications to MBP. We further show that MBP appears to be co-localized with actin filaments and microtubules in cultured oligodendrocytes, and also at the interface between actin filaments at the leading edge of membrane processes and microtubules behind them. Thus, MBP may also cross-link microtubules to actin filaments in vivo.Display Omitted► Myelin basic protein (MBP) binds microtubules (MTs) to lipid vesicles in vitro. ► Phosphorylation and Arg deimination decrease MBP-mediated tethering of MTs to membranes. ► MBP binds actin microfilaments (MFs) to MTs in vitro. ► MBP colocalizes with MFs and MTs in processes of cultured oligodendrocytes. ► MBP may act as a membrane and cytoskeletal scaffolding protein in vivo.

Keywords: Abbreviations; bC1; C1 isolated from bovine brain; bC1-P; bC1 phosphorylated; in vitro; by MAPK; C1; naturally occurring, least modified, most positively-charged component of 18.5; kDa MBP; C2; C3, C4, C5, C6, other naturally occurring charge components of MBP successively differing in charge by −; 1 due to various post-translational modifications; C8; least positively-charged component of MBP with 6 Arg replaced by citrulline; CaM; calmodulin; F-tubulin; fluorescein-labeled tubulin; GFP; green fluorescent protein; LUVs; large unilamellar vesicles; MAP; microtubule-associated protein; MAPK; mitogen-activated protein kinase; MARCKS; myristoylated alanine-rich C kinase substrate; MBP; myelin basic protein; OLs; oligodendrocytes; rmC8; His-tagged recombinant analogue of murine C8 with 6 Arg/Lys replaced by glutamine; PC; phosphatidylcholine; PG; phosphatidylglycerol; R-actin; rhodamine-labeled actin; RFP; red fluorescent protein; TEM; transmission electron microscopyIntrinsically disordered protein; Polyanion; Cytoskeleton; Liposome; Oligodendrocyte; Citrulline

On the miscibility of cardiolipin with 1,2-diacyl phosphoglycerides: Binary mixtures of dimyristoylphosphatidylethanolamine and tetramyristoylcardiolipin by Maria Frias; Matthew G.K. Benesch; Ruthven N.A.H. Lewis; Ronald N. McElhaney (pp. 774-783).

The thermotropic phase behavior and organization of model membranes composed of binary mixtures of the quadruple-chained, anionic phospholipid tetramyristoylcardiolipin (TMCL) with the double-chained zwitterionic phospholipid dimyristoylphosphatidylethanolamine (DMPE) were examined by a combination of differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. After equilibration at low temperature, DSC thermograms exhibited by binary mixtures of TMCL and DMPE containing <80mol DMPE exhibit a fairly energetic lower temperature endotherm and a highly energetic higher temperature endotherm. As the relative amount of TMCL in the mixture decreases, the temperature, enthalpy and cooperativity of the lower temperature endotherm also decreases and is not calorimetrically detectable when the TMCL content falls below 20mol%. In contrast, the temperature of the higher temperature endotherm increases as the proportion of TMCL decreases, but the enthalpy and cooperativity both decrease and the transition endotherms become multimodal. The FTIR spectroscopic results indicate that the lower temperature endotherm corresponds to a lamellar crystalline (L_c) to lamellar gel (L_β) phase transition and that the higher temperature transition involves the conversion of the L_β phase to the lamellar liquid-crystalline (L_α) phase. Moreover, the FTIR spectroscopic signatures observed at temperatures below the onset of the L_c/L_β phase transitions are consistent with the coexistence of structures akin to a TMCL-like L_c phase and the L_β phase, and with the relative amount of the TMCL-like L_c phase increasing progressively as the TMCL content of the mixture increases. These latter observations suggest that the TMCL and DMPE components of these mixtures are poorly miscible at temperatures below the L_β/L_α phase transition temperature. Poor miscibility of these two components is also suggested by the complexity of the DSC thermograms observed at the L_β/L_α phase transitions of these mixtures and with the complex relationship between their L_β/L_α phase transition temperatures and the composition of the mixture. Overall, our data suggests that TMCL and DMPE may be intrinsically poorly miscible across a broad composition range, notwithstanding the homogeneity of the fatty acid chains of the two components and the modest (~10°C) difference between their L_β/L_α phase transition temperatures.►Mixing of DMPE and TMCL is non-ideal over the composition range 5–95mol.%. ►Hydrocarbon chain-melting temperatures and mixture composition are poorly correlated. ►Chain-melting enthalpy is strongly correlated with the mol fraction of TMCL chains. ►Mixtures containing ≥10mol % TMCL form TMCL-like L_c phases upon cooling. ►TMCL inhibits the formation of DMPE-like L_c phases in DMPE-rich mixtures.

Keywords: Abbreviations; DMPE; dimyristoylphosphatidylethanolamine; CL; cardiolipin; PE; phosphatidylethanolamine; DMPG; dimyristoylphosphatidylglycerol; DMPC; dimyristoylphosphatidylcholine; TMCL; 1,2,-1′,2′-tetramyristoylcardiolipin; DSC; differential scanning calorimetry; IR; infrared; FTIR; Fourier-transform infrared; C=O; carbonyl; CH; ₂; methylene; L; _α; lamellar liquid-crystalline; L; _β; lamellar gel; L; _c; lamellar crystalline; T; _m; gel/liquid-crystalline phase transition temperatureCardiolipin; Phosphatidylethanolamine; Thermotropic phase behavior; Lipid bilayer; Differential scanning calorimetry; Infrared spectroscopy

Structure and dynamics of the lipid modifications of a transmembrane α-helical peptide determined by²H solid-state NMR spectroscopy by Anja Penk; Muller Matthias Müller; Holger A. Scheidt; Dieter Langosch; Daniel Huster (pp. 784-791).

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used²H and³¹P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the²H and³¹P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region.²H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant.► Transmembrane α-helical peptides with lipid chains attached were investigated. ► Lipid chains with 8 or more carbons fully insert into the membrane. ► No chain matching of those chains with the length of the host membrane was observed. ► Chains could exert a stabilizing effect on the peptide.

Keywords: LV peptide; Acylation; Lipid modification; Order parameter

Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses by Olena M. Nesin; Olga N. Pakhomova; Shu Xiao; Andrei G. Pakhomov (pp. 792-801).

Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in the cell membrane, followed by cell volume changes due to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by either fifty 60-ns EP (~13 kV/cm) or five, 600-ns EP (~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with polyethylene glycols and sugars. Such replacement reduced cell swelling or resulted in transient or sustained cell shrinking in response to EP. depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as estimated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.► We compared membrane nanopores produced by 60- and 600-ns electric pulses. ► Nanopore opening caused cell volume changes and propidium uptake. ► Nanopore size was probed by different molecular size osmolates. ► Unexpectedly, we found that pore size profiles after 60- and 600-ns pulses are similar. ► However, 600-ns pulses opened a small fraction of larger pores.

Keywords: Abbreviations; EP; electric pulses; nsEP; nanosecond-duration electric pulses; PEGs; polyethylene glycols; Pr; propidiumMembrane permeability; Colloid osmotic effect; Pore size; Nanopores; Nanosecond pulses; Electroporation

Homology modeling of human CCR5 and analysis of its binding properties through molecular docking and molecular dynamics simulation by Mohsen Shahlaei; Armin Madadkar-Sobhani; Karim Mahnam; Afshin Fassihi; Lotfollah Saghaie; Mahboubeh Mansourian (pp. 802-817).

In this study, homology modeling, molecular docking and molecular dynamics simulation were performed to explore structural features and binding mechanism of some inhibitors of chemokine receptor type 5 (CCR5), and to construct a model for designing new CCR5 inhibitors for preventing HIV attachment to the host cell. A homology modeling procedure was employed to construct a 3D model of CCR5. For this procedure, the X-ray crystal structure of bovine rhodopsin (1F88A) at 2.80Å resolution was used as template. After inserting the constructed model into a hydrated lipid bilayer, a 20ns molecular dynamics (MD) simulation was performed on the whole system. After reaching the equilibrium, twenty-four CCR5 inhibitors were docked in the active site of the obtained model. The binding models of the investigated antagonists indicate the mechanism of binding of the studied compounds to the CCR5 obviously. Moreover, 3D pictures of inhibitor–protein complex provided precious data regarding the binding orientation of each antagonist into the active site of this protein. One additional 20ns MD simulation was performed on the initial structure of the CCR5–ligand 21 complex, resulted from the previous docking calculations, embedded in a hydrated POPE bilayer to explore the effects of the presence of lipid bilayer in the vicinity of CCR5–ligand complex. This article is part of a Special Issue entitled Protein translocation across or insertion into membranesIn this work, homology modeling, docking and molecular dynamics simulation were performed to explore binding mechanism of some inhibitors of the CCR5.Display Omitted► Construction of 3D model of CCR5 as a membrane protein. ► Molecular dynamics simulation study of CCR5 in the presence of lipid membrane. ► Exploring of CCR5 function with considering lipid membrane effects explicitly. ► Exploring mechanism of action of some antagonists in active site of CCR5.

Keywords: CCR5; Homology modeling; Molecular docking; Molecular dynamics simulation

An NMR database for simulations of membrane dynamics by Avigdor Leftin; Michael F. Brown (pp. 818-839).

Computational methods are powerful in capturing the results of experimental studies in terms of force fields that both explain and predict biological structures. Validation of molecular simulations requires comparison with experimental data to test and confirm computational predictions. Here we report a comprehensive database of NMR results for membrane phospholipids with interpretations intended to be accessible by non-NMR specialists. Experimental¹³C–¹H and²H NMR segmental order parameters ( S_CH or S_CD) and spin-lattice (Zeeman) relaxation times ( T1_Z) are summarized in convenient tabular form for various saturated, unsaturated, and biological membrane phospholipids. Segmental order parameters give direct information about bilayer structural properties, including the area per lipid and volumetric hydrocarbon thickness. In addition, relaxation rates provide complementary information about molecular dynamics. Particular attention is paid to the magnetic field dependence (frequency dispersion) of the NMR relaxation rates in terms of various simplified power laws. Model-free reduction of the T1_Z studies in terms of a power-law formalism shows that the relaxation rates for saturated phosphatidylcholines follow a single frequency-dispersive trend within the MHz regime. We show how analytical models can guide the continued development of atomistic and coarse-grained force fields. Our interpretation suggests that lipid diffusion and collective order fluctuations are implicitly governed by the viscoelastic nature of the liquid-crystalline ensemble. Collective bilayer excitations are emergent over mesoscopic length scales that fall between the molecular and bilayer dimensions, and are important for lipid organization and lipid-protein interactions. Future conceptual advances and theoretical reductions will foster understanding of biomembrane structural dynamics through a synergy of NMR measurements and molecular simulations.► Comprehensive NMR database for saturated, unsaturated, biomembrane phospholipids. ► Extensive¹³C and² H NMR order parameter and NMR relaxation time tabulation. ► Analytic models as a guide for simulations of phospholipid membrane dynamics. ► Model-free interpretation of continuous quasi-elastic bilayer mode distributions. ► Hierarchical properties of lipid membranes emergent on mesoscopic length scale.

Keywords: Abbreviations; Chol; cholesterol; CPMG; Carr-Purcell-Meiboom-Gill; CSA; chemical shift anisotropy; DDPC; 1,2-didocosahexaenoyl-; sn; -glycero-3-phosphocholine; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphocholine; DLPC-; d; ₄₆; 1,2-diperdeuteriolauroyl-; sn; -glycero-3-phosphocholine; D; methylene travel; D; _M; maximum methylene travel; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPC-; d; ₅₄; 1,2-diperdeuteriomyristoyl-; sn; -glycero-3-phosphocholine; DMPE; 1,2-dimyristoyl-; sn; -glycero-3-phosphoethanolamine; DMPE-; d; ₂₇; 1-myristoyl-2-perdeuteriomyristoyl-; sn; -glycero-3-phosphoethanolamine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPC-; d; ₆₂; 1,2-diperdeuteriopalmitoyl-; sn; -glycero-3-phosphocholine; DROSS; dipolar recoupling with shape and sign preservation; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; DSPC-; d; ₇₂; 1,2-diperdeuteriostearoyl-; sn; -glycero-3-phosphocholine; GalCer; galactosylceramide; GlcCer; glucosylceramide; H; _II; inverted hexagonal phase; MAS; magic angle spinning; MD; molecular dynamics; NMR; nuclear magnetic resonance; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PLPE-; d; ₃₁; 1-perdeuteriopalmitoyl-2-linoleoyl-; sn; -glycero-3-phosphoethanolamine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PS; phosphatidylserine; PSM; N-palmitoyl sphingomyelin; PI; phosphatidylinositol; SAXS; small-angle X-ray scattering; SLF; separated local-fieldCholesterol; Lipid bilayer; Lipid–protein interactions; Molecular dynamics; Nuclear spin relaxation; Order parameter; Phospholipid; Rafts; Solid-state NMR

An NMR database for simulations of membrane dynamics by Avigdor Leftin; Michael F. Brown (pp. 818-839).

Special section on “Protein translocation across or insertion into membranes” by Doron Rapaport (pp. 840-840).

Early targeting events during membrane protein biogenesis in Escherichia coli by Eitan Bibi (pp. 841-850).

All living cells have co-translational pathways for targeting membrane proteins. Co-translation pathways for secretory proteins also exist but mostly in eukaryotes. Unlike secretory proteins, the biosynthetic pathway of most membrane proteins is conserved through evolution and these proteins are usually synthesized by membrane-bound ribosomes. Translation on the membrane requires that both the ribosomes and the mRNAs be properly localized. Theoretically, this can be achieved by several means. (i) The current view is that the targeting of cytosolic mRNA-ribosome-nascent chain complexes (RNCs) to the membrane is initiated by information in the emerging hydrophobic nascent polypeptides. (ii) The alternative model suggests that ribosomes may be targeted to the membrane also constitutively, whereas the appropriate mRNAs may be carried on small ribosomal subunits or targeted by other cellular factors to the membrane-bound ribosomes. Importantly, the available experimental data do not rule out the possibility that cells may also utilize both pathways in parallel. In any case, it is well documented that a major player in the targeting pathway is the signal recognition particle (SRP) system composed of the SRP and its receptor (SR). Although the functional core of the SRP system is evolutionarily conserved, its composition and biological practice come with different flavors in various organisms. This review is dedicated mainly to the Escherichia ( E.) coli SRP, where the biochemical and structural properties of components of the SRP system have been relatively characterized, yielding essential information about various aspects of the pathway. In addition, several cellular interactions of the SRP and its receptor have been described in E. coli, providing insights into their spatial function. Collectively, these in vitro studies have led to the current view of the targeting pathway [see (i) above]. Interestingly, however, in vivo studies of the role of the SRP and its receptor, with emphasis on the temporal progress of the pathway, elicited an alternative hypothesis [see (ii) above]. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Signal recognition particle; SRP; SRP-receptor; SRP–RNA; Ffh; FtsY; 4.5S RNA; Escherichia coli; Integral membrane protein; Ribosome; mRNA targeting

Early targeting events during membrane protein biogenesis in Escherichia coli by Eitan Bibi (pp. 841-850).

Keywords: Signal recognition particle; SRP; SRP-receptor; SRP–RNA; Ffh; FtsY; 4.5S RNA; Escherichia coli; Integral membrane protein; Ribosome; mRNA targeting

The Sec translocase by David J.F. du Plessis; Nico Nouwen; Arnold J.M. Driessen (pp. 851-865).

The vast majority of proteins trafficking across or into the bacterial cytoplasmic membrane occur via the translocon. The translocon consists of the SecYEG complex that forms an evolutionarily conserved heterotrimeric protein-conducting membrane channel that functions in conjunction with a variety of ancillary proteins. For posttranslational protein translocation, the translocon interacts with the cytosolic motor protein SecA that drives the ATP-dependent stepwise translocation of unfolded polypeptides across the membrane. For the cotranslational integration of membrane proteins, the translocon interacts with ribosome-nascent chain complexes and membrane insertion is coupled to polypeptide chain elongation at the ribosome. These processes are assisted by the YidC and SecDF(yajC) complex that transiently interacts with the translocon. This review summarizes our current understanding of the structure–function relationship of the translocon and its interactions with ancillary components during protein translocation and membrane protein insertion. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.►Structure-function relationship of the protein-conducting channel, the translocon. ►Overview of the targeting pathways of (membrane) proteins to the translocon. ►Functional description of the subunits of the translocon.

Keywords: SecA; SecYEG; YidC; Secretion; Membrane protein

The Sec translocase by David J.F. du Plessis; Nico Nouwen; Arnold J.M. Driessen (pp. 851-865).

Keywords: SecA; SecYEG; YidC; Secretion; Membrane protein

Inserting membrane proteins: The YidC/Oxa1/Alb3 machinery in bacteria, mitochondria, and chloroplasts by Peng Wang; Ross E. Dalbey (pp. 866-875).

The evolutionarily conserved YidC/Oxa1p/Alb3 family of proteins plays important roles in the membrane biogenesis in bacteria, mitochondria, and chloroplasts. The members in this family function as novel membrane protein insertases, chaperones, and assembly factors for transmembrane proteins, including energy transduction complexes localized in the bacterial and mitochondrial inner membrane, and in the chloroplast thylakoid membrane. In this review, we will present recent progress with this class of proteins in membrane protein biogenesis and discuss the structure/function relationships. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.►The YidC insertase promotes membrane insertion of a subset of proteins into the cytoplasmic membrane in bacteria. ►The YidC insertase can function as a chaperone and can facilitate the folding of membrane proteins. ►Oxa1 plays a crucial role in the assembly of the mitochodrial inner membrane proteins cytochrome c oxidase and F1Fo ATP synthase. ►Alb3 mediates the integration of SRP-dependent light-harvesting chlorophyll a, b binding proteins into the chloroplast thylakoid membrane.

Keywords: Membrane protein insertion; Protein export; Membrane targeting; YidC; Oxa1; Alb3

Inserting membrane proteins: The YidC/Oxa1/Alb3 machinery in bacteria, mitochondria, and chloroplasts by Peng Wang; Ross E. Dalbey (pp. 866-875).

Keywords: Membrane protein insertion; Protein export; Membrane targeting; YidC; Oxa1; Alb3

Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria by Colin Robinson; Cristina F.R.O. Matos; Daniel Beck; Chao Ren; Janna Lawrence; Nishi Vasisht; Sharon Mendel (pp. 876-884).

The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler “minimalist” Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated “proofreading” activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Tat; Twin-arginine; Protein transport; Signal peptide; Membrane protein

Crossing the membrane in Archaea, the third domain of life by Doron Calo; Jerry Eichler (pp. 885-891).

Many of the recent advancements in the field of protein translocation, particularly from the structural perspective, have relied on Archaea. For instance, the solved structures of the translocon from the methanoarchaeon Methanocaldococcus jannaschii of the ribosomal large subunit from the haloarchaeon Haloarcula marismortui and of components of the SRP pathway from several archaeal species have provided novel insight into various aspects of the translocation event. Given the major contribution that Archaea have made to our understanding of how proteins enter and traverse membranes, it is surprising that relatively little is known of protein translocation in Archaea in comparison to the well-defined translocation pathways of Eukarya and Bacteria. What is known, however, points to archaeal translocation as comprising a mosaic of eukaryal and bacterial traits together with aspects of the process seemingly unique to this, the third domain of life. Here, current understanding of archaeal protein translocation is considered. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Archaea; Protein translocation; Signal recognition particle; Translocon; Signal peptidase; Twin arginine translocation

Crossing the membrane in Archaea, the third domain of life by Doron Calo; Jerry Eichler (pp. 885-891).

Keywords: Archaea; Protein translocation; Signal recognition particle; Translocon; Signal peptidase; Twin arginine translocation

Protein import machineries of peroxisomes by Rucktaschel Robert Rucktäschel; Wolfgang Girzalsky; Ralf Erdmann (pp. 892-900).

Peroxisomes are a class of structurally and functionally related organelles present in almost all eukaryotic cells. The importance of peroxisomes for human life is highlighted by severe inherited diseases which are caused by defects of peroxins, encoded by PEX genes. To date 32 peroxins are known to be involved in different aspects of peroxisome biogenesis. This review addresses two of these aspects, the translocation of soluble proteins into the peroxisomal matrix and the biogenesis of the peroxisomal membrane. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Abbreviations; AAA; ATPase associated with various cellular activities; PTS; peroxisomal targeting signal; RING; really interesting new gene; Ub; ubiquitinPeroxisome biogenesis; Protein targeting; PEX; Peroxin

Protein import machineries of peroxisomes by Rucktaschel Robert Rucktäschel; Wolfgang Girzalsky; Ralf Erdmann (pp. 892-900).

Protein import into chloroplasts—How chaperones feature into the game by Serena Schwenkert; Jürgen Soll; Bolter Bettina Bölter (pp. 901-911).

Chloroplasts originated from an endosymbiotic event, in which an ancestral photosynthetic cyanobacterium was engulfed by a mitochondriate eukaryotic host cell. During evolution, the endosymbiont lost its autonomy by means of a massive transfer of genetic information from the prokaryotic genome to the host nucleus. Consequently, the development of protein import machineries became necessary for the relocation of proteins that are now nuclear-encoded and synthesized in the cytosol but destined for the chloroplast. Organelle biogenesis and maintenance requires a tight coordination of transcription, translation and protein import between the host cell and the organelle. This review focuses on the translocation complexes in the outer and inner envelope membrane with a special emphasis on the role of molecular chaperones. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Chloroplasts; Endosymbiosis; Protein import; Toc/Tic complexes; Redox regulation; Thiol-regulation; Hsp90; Hsp70; Hsp93

Protein import into chloroplasts—How chaperones feature into the game by Serena Schwenkert; Jürgen Soll; Bolter Bettina Bölter (pp. 901-911).

Keywords: Chloroplasts; Endosymbiosis; Protein import; Toc/Tic complexes; Redox regulation; Thiol-regulation; Hsp90; Hsp70; Hsp93

Protein translocation across the ER membrane by Richard Zimmermann; Susanne Eyrisch; Mazen Ahmad; Volkhard Helms (pp. 912-924).

Protein translocation into the endoplasmic reticulum (ER) is the first and decisive step in the biogenesis of most extracellular and many soluble organelle proteins in eukaryotic cells. It is mechanistically related to protein export from eubacteria and archaea and to the integration of newly synthesized membrane proteins into the ER membrane and the plasma membranes of eubacteria and archaea (with the exception of tail anchored membrane proteins). Typically, protein translocation into the ER involves cleavable amino terminal signal peptides in precursor proteins and sophisticated transport machinery components in the cytosol, the ER membrane, and the ER lumen. Depending on the hydrophobicity and/or overall amino acid content of the precursor protein, transport can occur co- or posttranslationally. The respective mechanism determines the requirements for certain cytosolic transport components. The two mechanisms merge at the level of the ER membrane, specifically, at the heterotrimeric Sec61 complex present in the membrane. The Sec61 complex provides a signal peptide recognition site and forms a polypeptide conducting channel. Apparently, the Sec61 complex is gated by various ligands, such as signal peptides of the transport substrates, ribosomes (in cotranslational transport), and the ER lumenal molecular chaperone, BiP. Binding of BiP to the incoming polypeptide contributes to efficiency and unidirectionality of transport. Recent insights into the structure of the Sec61 complex and the comparison of the transport mechanisms and machineries in the yeast Saccharomyces cerevisiae, the human parasite Trypanosoma brucei, and mammals have various important mechanistic as well as potential medical implications. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Abbreviations; BiP; immunoglobulin heavy chain binding protein; EM; electron microscopy; ER; endoplasmic reticulum; ERj; ER resident J-domain protein; GPI; glycosylphosphatidylinositol; Grp; glucose regulated protein; Hsp; heat shock protein; NAC; nascent chain associated complex; NEF; nucleotide exchange factor; OST; oligisaccharyl transferase; RAMP; ribosome associated membrane protein; Sec; protein or complex that is involved in protein secretion; SPC; signal peptidase complex; SRP; signal recognition particle; SR; SRP receptorDriving force; Endoplasmic reticulum; Membrane insertion; Molecular chaperones; Protein translocation; Structural analysis

Protein translocation across the ER membrane by Richard Zimmermann; Susanne Eyrisch; Mazen Ahmad; Volkhard Helms (pp. 912-924).

Protein dislocation from the ER by Katrin Bagola; Martin Mehnert; Ernst Jarosch; Thomas Sommer (pp. 925-936).

Protein folding within the endoplasmic reticulum (ER) of eukaryotic cells is erroneous and often results in the formation of terminally malfolded species. A quality control system retards such molecules in the ER and eventually initiates their dislocation into the cytosol for proteolysis by 26S proteasomes. This process is termed ER associated protein degradation (ERAD). The spatial separation of ER based quality control and cytosolic proteolysis poses the need for a machinery that promotes the extraction of substrates from the ER. Due to the heterogeneous nature of the client proteins this transport system displays several unique features. Selective recognition of ERAD substrates does not involve transferable transport signals in the primary sequence and thus must follow other principles than established for proteins designated for the import into organelles. Moreover, an ER dislcocation system must be capable to ship polypeptides, which may be at least partly folded and are in most cases covalently modified with bulky and hydrophilic glycans, through a membrane without disrupting the integrity of the ER. In this review we present current ideas on the highly dynamic and flexible nature of the dislocation apparatus and speculate on the mechanism that removes aberrant polypeptides from the ER in the course of ERAD. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.► Misfolded proteins of the secretory pathway are degraded by cytosolic proteasomes. ► Proteolysis of such molecules is preceded by their removal from the endoplasmic reticulum (ER). ► Key players in this process are multimeric ubiquitin ligases that are integrated into the ER membrane. ► These ligase complexes partake in the selection and polyubiquitylation of substrates and may also contribute to the formation of dynamic channels for protein export.

Keywords: ERAD; Membrane transport; Ubiquitin ligase; Protein quality control

Protein dislocation from the ER by Katrin Bagola; Martin Mehnert; Ernst Jarosch; Thomas Sommer (pp. 925-936).

Keywords: ERAD; Membrane transport; Ubiquitin ligase; Protein quality control

Targeting pathways of C-tail-anchored proteins by Nica Borgese; Elisa Fasana (pp. 937-946).

A large group of diverse, functionally important, and differently localized transmembrane proteins, share a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. The C-terminal membrane anchor of these tail-anchored (TA) proteins generally represents the sole targeting determinant, and becomes available to targeting factors only after release of the finished polypeptide from the ribosome. Hence, TA proteins do not have a chance to interact co-translationally with Signal Recognition Particle and are delivered post-translationally to all target membranes, including the ER. Recent work has demonstrated the existence of different biogenetic pathways for TA proteins. Notably, some are able to efficiently translocate their C-terminus across protein-free bilayers without the participation of any membrane or cytosolic protein, while others require assistance from cytosolic chaperones and membrane receptors. In this review, we summarize current knowledge on the different insertion pathways, with emphasis on a recently discovered chaperone system that operates in fungi as well as in higher eukaryotes to deliver TA proteins to the ER (called Guided Entry of Tail-anchored Proteins (Get) system and Transmembrane Recognition Complex (TRC), in yeast and mammals, respectively). We suggest that the final insertion step of TA proteins does not require membrane proteins, but that different competing chaperone systems ensure precise delivery to defined targets while preventing inappropriate insertion into otherwise permissive bilayers. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Abbreviations; b5; cytochrome b5; COE; chloroplast outer envelope; ER; Endoplasmic Reticulum; Get; Guided Entry of Tail-anchored Proteins; Hsc; Heat Shock Cognate Protein; Hsp; Heat Shock Protein; OMM; outer mitochondrial membrane; RRL; rabbit reticulocyte lysate; SNARE; soluble; N; -ethylmaleimide-sensitive factor attachment protein receptor; SRP; Signal Recognition Particle; Syb; synaptobrevin; TMD; transmembrane domain; TOM; translocase of the outer membrane; TRC40; Transmembrane Recognition Complex subunit of 40; kDa; Ubl; Ubiquitin like domainTRC40/Get3; Unassisted insertion; Chaperone

Targeting pathways of C-tail-anchored proteins by Nica Borgese; Elisa Fasana (pp. 937-946).

Minor modifications and major adaptations: The evolution of molecular machines driving mitochondrial protein import by Victoria Hewitt; Felicity Alcock; Trevor Lithgow (pp. 947-954).

Bacterial endosymbionts gave rise to mitochondria in a process that depended on the acquisition of protein import pathways. Modification and in some cases major re-tooling of the endosymbiont's cellular machinery produced these pathways, establishing mitochondria as organelles common to all eukaryotic cells. The legacy of this evolutionary tinkering can be seen in the homologies and structural similarities between mitochondrial protein import machinery and modern day bacterial proteins. Comparative analysis of these systems is revealing both possible routes for the evolution of the mitochondrial membrane translocases and a greater understanding of the mechanisms behind mitochondrial protein import. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Mitochondria; Evolution; Protein translocation; Alpha-proteobacteria; Membranes

Minor modifications and major adaptations: The evolution of molecular machines driving mitochondrial protein import by Victoria Hewitt; Felicity Alcock; Trevor Lithgow (pp. 947-954).

Keywords: Mitochondria; Evolution; Protein translocation; Alpha-proteobacteria; Membranes

Structural insight into the mitochondrial protein import system by Toshiya Endo; Koji Yamano; Shin Kawano (pp. 955-970).

Mitochondrial functions rely on precise and efficient transport of 1000–1500 different mitochondrial proteins from the cytosol to appropriate mitochondrial subcompartments. Those mitochondrial protein transport processes are mediated by the dedicated mitochondrial protein import system comprised of translocators in the outer and inner mitochondrial membranes and soluble factors in the cytosol, intermembrane space, and matrix. In the last decade, high-resolution structures of many of the components of the mitochondrial protein import machineries have become available, which has significantly advanced our understanding of the molecular mechanisms of mitochondrial protein transport. Here we review the currently available high-resolution structures of the components of the mitochondrial protein import machineries that afford structural and mechanistic insight into how the mitochondrial import system works. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Protein import; Mitochondria; Translocator; Receptor; X-ray structure; NMR structure

Structural insight into the mitochondrial protein import system by Toshiya Endo; Koji Yamano; Shin Kawano (pp. 955-970).

Keywords: Protein import; Mitochondria; Translocator; Receptor; X-ray structure; NMR structure

Multiple pathways in the integration of proteins into the mitochondrial outer membrane by Jovana Dukanovic; Doron Rapaport (pp. 971-980).

Proteins residing in the mitochondrial outer membrane facilitate various interactions between the organelle and the rest of the eukaryotic cell. All these proteins are encoded in the nucleus and synthesized on cytosolic ribosomes. Thus, they have to bear appropriate signals that ensure both their correct import into the organelle and their ability to acquire different topologies in the lipid bilayer. None of these proteins contain a canonical cleavable N-terminal presequence. Rather, the targeting and sorting signals can be found at their termini or in internal structural elements. In this review, we summarize the current knowledge regarding the diverse molecular mechanisms by which mitochondrial outer membrane proteins are specifically targeted to the organelle and inserted into the target membrane. Recognition events in the diverse pathways and the driving force for the various stages of this process will be discussed. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Mitochondria; Outer membrane; Beta-barrel proteins; Tail-anchored proteins; TOB complex; TOM complex

Multiple pathways in the integration of proteins into the mitochondrial outer membrane by Jovana Dukanovic; Doron Rapaport (pp. 971-980).

Keywords: Mitochondria; Outer membrane; Beta-barrel proteins; Tail-anchored proteins; TOB complex; TOM complex

Oxidation-driven protein import into mitochondria: Insights and blind spots by Jan Riemer; Manuel Fischer; Johannes M. Herrmann (pp. 981-989).

The intermembrane space of mitochondria contains a dedicated machinery for the introduction of disulfide bonds into proteins. In this case, oxidative protein folding is believed to drive the vectorial translocation of polypeptides after their synthesis in the cytosol across the mitochondrial outer membrane. Substrates of this system are recognized by a hydrophobic binding cleft of the oxidoreductase Mia40 which converts them into an oxidized stably folded conformation. Mia40 is maintained in an oxidized, active conformation by the sulfhydryl oxidase Erv1, a homodimeric flavoenzyme, which can form disulfide bonds de novo. Erv1 passes electrons on to cytochrome c and further to the respiratory chain. The components of this system, their structures and the mechanisms of disulfide bond formation were analyzed only very recently. This review discusses our knowledge about this system as well as open questions which still wait to be addressed. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Abbreviations; Ccs1; copper chaperone for Sod1; GSH; reduced glutathione; IMS; intermembrane space; ITS; IMS-targeting signal; MISS; mitochondria IMS-sorting signal; MTS; matrix targeting signal; Sod1; superoxide dismutase 1; TIM; translocase of the inner membrane; TOM; translocase of the outer membraneGlutathione; Erv1; Mia40; Mitochondria; Protein folding; Protein oxidation

Oxidation-driven protein import into mitochondria: Insights and blind spots by Jan Riemer; Manuel Fischer; Johannes M. Herrmann (pp. 981-989).

Understanding the molecular mechanism of protein translocation across the mitochondrial inner membrane: Still a long way to go by Milit Marom; Abdussalam Azem; Dejana Mokranjac (pp. 990-1001).

In order to reach the final place of their function, approximately half of the proteins in any eukaryotic cell have to be transported across or into one of the membranes in the cell. In this article, we present an overview of our current knowledge concerning the structural properties of the TIM23 complex and their relationship with the molecular mechanism of protein transport across the mitochondrial inner membrane. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: TOM; TIM23; Tim44; mtHsp70; Translocation motor

Understanding the molecular mechanism of protein translocation across the mitochondrial inner membrane: Still a long way to go by Milit Marom; Abdussalam Azem; Dejana Mokranjac (pp. 990-1001).

Keywords: TOM; TIM23; Tim44; mtHsp70; Translocation motor

Mitochondrial protein import machineries and lipids: A functional connection by Natalia Gebert; Michael T. Ryan; Nikolaus Pfanner; Nils Wiedemann; Diana Stojanovski (pp. 1002-1011).

Protein trafficking and translocation are essential processes in even the simplest living cells. The compartmentalisation within eukaryotic cells places a very high demand on the fidelity of protein trafficking and translocation, since a large percentage of the cell's protein complement is inserted into, or translocated across membranes. Indeed, most mitochondrial proteins are imported from the cytosol into the organelle and reach their final destination with the assistance of versatile translocation machineries. The first components involved in mitochondrial protein import were identified about 20years ago and over the last two decades many new factors and machineries have been brought to light. However, in spite of these discoveries we still have much to explore regarding the molecular mechanisms that distinguish the different mitochondrial import pathways. In particular, an open question that requires deeper exploration is the role of lipids and lipid modifying enzymes in this process. Mitochondrial biogenesis requires the coordinated synthesis and import of both proteins and phospholipids, however, these have typically been considered as distinct research fields. Recent findings have placed phospholipids at the forefront of research dealing with mitochondrial biogenesis, in particular their role in the regulation of mitochondrial transport machineries. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Abbreviations; CDP-DAG; CDP-diacylglycerol; CL; cardiolipin; ER; endoplasmic reticulum; MDM; mitochondrial distribution and morphology; MIA; mitochondrial intermembrane space assembly; MLCL; monolysocardiolipin; Mmm1; maintenance of mitochondrial morphology; mtHsp70; mitochondrial heat shock protein 70; PA; phosphatidic acid; PAM; presequence translocase-associated motor; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PGP; phosphatidylglycerol phosphate; PI; phosphatidylinositol; PS; phosphatidylserine; SAM; sorting and assembly machinery; Tam41; translocator assembly and maintenance protein 41; TIM22; carrier translocase of inner membrane; TIM23; presequence translocase of inner membrane; TOM; translocase of outer membraneMitochondria; Protein import; Cardiolipin; Lipids

Mitochondrial protein import machineries and lipids: A functional connection by Natalia Gebert; Michael T. Ryan; Nikolaus Pfanner; Nils Wiedemann; Diana Stojanovski (pp. 1002-1011).

Dual targeting of mitochondrial proteins: Mechanism, regulation and function by Ohad Yogev; Ophry Pines (pp. 1012-1020).

One solution found in evolution to increase the number of cellular functions, without increasing the number of genes, is distribution of single gene products to more than one cellular compartment. It is well documented that in eukaryotic cells, molecules of one protein can be located in several subcellular locations, a phenomenon termed dual targeting, dual localization, or dual distribution. The differently localized proteins are coined in this review “echoforms” indicating repetitious forms of the same protein (echo in Greek denotes repetition) distinctly placed in the cell. This term replaces the term to “isoproteins” or “isoenzymes” which are reserved for proteins with the same activity but different amino acid sequences. Echoforms are identical or nearly identical, even though, as referred to in this review may, in some cases, surprisingly have a totally different function in the different compartments. With regard to mitochondria, our operational definition of dual targeted proteins refers to situations in which one of the echoforms is translocated through/into a mitochondrial membrane.In this review we ask how, when and why mitochondrial proteins are dual localized in the cell. We describe mechanisms of dual targeting of proteins between mitochondria and other compartments of the eukaryotic cell. In particular, we have paid attention to situations in which dual localization is regulated in time, location or function. In addition, we have attempted to provide a broader view concerning the phenomenon of dual localization of proteins by looking at mechanisms that are beyond our simple definition of dual targeting. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Keywords: Dual targeting; Dual localization; Mitochondria; Targeting signal; Protein translocation; Organelle

Dual targeting of mitochondrial proteins: Mechanism, regulation and function by Ohad Yogev; Ophry Pines (pp. 1012-1020).

Keywords: Dual targeting; Dual localization; Mitochondria; Targeting signal; Protein translocation; Organelle

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BBA - Biomembranes (v.1808, #3)