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BBA - Biomembranes (v.1808, #4)

Editorial Board (pp. i).

A study of the membrane association and regulatory effect of the phospholemman cytoplasmic domain by Eleri Hughes; Christopher A.P. Whittaker; Igor L. Barsukov; Mikael Esmann; David A. Middleton (pp. 1021-1031).

Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K⁺ and Na⁺ and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM_38–72) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM_38–72 associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM_38–72 also lowers the maximal velocity of ATP hydrolysis ( V_max) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V_max. These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.► The phospholemman cytoplasmic domain (PCD) interacts weakly with zwitterionic phospholipids. ► Phosphorylation of PCD at S68 lowers its phospholipid membrane affinity. ► PCD lowers the maximal rate of ATP hydrolysis by Na,K-ATPase. ► The inhibitory effect of PCD is partially relieved by phosphorylation at S68. ► The physical and functional properties of CHIF and Mat-8 cytoplasmic domains differ from PCD.

Keywords: Abbreviations; PLM; phospholemman (FXYD1); PLM; _38–72; a peptide representing residues 38–72 of phospholemman; Mat-8 (FXYD3); mammary tumor protein; CHIF (FXYD4); corticosteroid induced factor; REDOR; rotational-echo double-resonance; SSNMR; solid-state NMR; ITC; isothermal titration calorimetry; NKA; Na; ⁺; ,K; ⁺; -ATPase; NCX; Na; ⁺; /Ca; ²⁺; exchanger; DMPC; dimyristoylphosphatidylcholine; DOPC; dioleoylphosphatidylcholine; DOPG; dimyristoylphosphatidylglycerol; DOPS; dioleoylphosphatidylserineFXYD; CHIF; Mat-8; Circular dichroism; Isothermal titration calorimetry; Solid-state NMR

Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein by Ana J. Vecino; Igor de la Arada; Rosa L. Segura; Goni Félix M. Goñi; Fernando de la Cruz; José L.R. Arrondo; Itziar Alkorta (pp. 1032-1039).

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim. Biophys. Acta 1798(11), 2160–2169 (2010)] that TrwB can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for ATP. In the present contribution we examine the structural effects of membrane insertion on TrwB, by comparing the protein in reconstituted form and in the form of protein/lipid/detergent mixed micelles. TrwB was reconstituted in PE:PG:CL (76.3:19.6:4.1mol ratio) with a final 99:1 lipid:protein mol ratio. This lipid mixture is intended to mimic the bacterial inner membrane composition, and allows a more efficient reconstitution than other lipid mixtures tested. The studies have been carried out mainly using infrared spectroscopy, because this technique provides simultaneously information on both the lipid and protein membrane components. Membrane reconstitution of TrwB is accompanied by a decrease in β-sheet contents and an increase in β-strand structures, probably related to protein–protein contacts in the bilayer. The predominant α-helical component remains unchanged. The bilayer-embedded protein becomes thermally more stable, and also more resistant to trypsin digestion. The properties of the bilayer lipids are also modified in the presence of TrwB, the phospholipid acyl chains are slightly ordered, and the phosphate groups at the interface become more accessible to water. In addition, we observe that the protein thermal denaturation affects the lipid thermal transition profile.► The α-helical component of TrwB remains unchanged when inserted into the bilayer. ► Incorporation of TrwB into lipid bilayers causes an increase in β-strand structures. ► β-strand structures are probably related to protein–protein contacts in the bilayer. ► TrwB adopts a more stable conformation when reconstituted into liposomes. ► Incorporation of TrwB into lipid bilayers causes an increase in lipid chain order.

Keywords: Abbreviations; T4CP; type IV coupling protein; TMD; transmembrane domain; IR; infrared spectroscopy; TNP-ATP; 2′(or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; CL; cardiolipin; OG; n-octyl β-; d; -glucopyranoside; DDM; n-Dodecyl-β-; d; -maltosideMembrane protein; Bacterial conjugation; Conjugative coupling protein; Protein reconstitution; R388; IR spectroscopy

Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins by Siti Azma Jusoh; Volkhard Helms (pp. 1040-1049).

Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix–helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys–Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1–E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.► Microsolvation of bridging ion pairs in transmembrane helix dimers. ► Microsolvation in membrane core stabilizes ion pairs and H-bonds between TM helices. ► Position of charged/polar amino acids affects the degree of their hydration. ► Buried waters exchange with bulk waters on timescales of tens of nanoseconds. ► Similar findings for H-segment dimers and for E1–E2 dimers from HCV.

Keywords: Abbreviations; TM; transmembrane; DMPC; dimyristoyl-phosphatidylcholine; DPPC; dipalmitoyl-phosphatidylcholineMolecular dynamics simulation; Charged amino acids; Heterodimerization; Hepatitis C; Envelope glycoproteins; Solvation; Membrane proteins; Flaviviridae; Dengue; West Nile; Japanese encephalitis; Bovine viral diarrhea; Ion-pair

Endocannabinoids and diacylglycerol kinase activity by Arpita Gantayet; Januvi Jegatheswaran; Gowtham Jayakumaran; Matthew K. Topham; Richard M. Epand (pp. 1050-1053).

Mammalian diacylglycerol kinases are a family of enzymes that catalyze the phosphorylation of diacylglycerol to produce phosphatidic acid. The extent of interaction of these enzymes with monoacylglycerols is the focus of the present study. Because of the structural relationship between mono- and diacylglycerols, one might expect the monoacylglycerols to be either substrates or inhibitors of diacylglycerol kinases. This would have some consequence to lipid metabolism. One of the lipid metabolites that would be affected is 2-arachidonoyl glycerol, which is an endogenous ligand for the CB1 cannabinoid receptor. We determined if the monoglycerides 2-arachidonoyl glycerol or 2-oleoyl glycerol affected diacylglycerol kinase activity. We found that 2-arachidonoyl glycerol is a very poor substrate for either the epsilon or the zeta isoforms of diacylglycerol kinases. Moreover, 2-arachidonoyl glycerol is an inhibitor for both of these diacylglycerol kinase isoforms. 2-oleoyl glycerol is also a poor substrate for these two isoforms of diacylglycerol kinases. As an inhibitor, 2-oleoyl glycerol inhibits diacylglycerol kinase ε less than does 2-arachidonoyl glycerol, while for diacylglycerol kinase ζ, these two monoglycerides have similar inhibitory potency. These results have implications for the known role of diacylglycerol kinase ε in neuronal function and in epilepsy since the action of this enzyme will remove 1-stearoyl-2-arachidonoylglycerol, the precursor of the endocannabinoid 2-arachidonoyl glycerol.►Specificity of diacylglycerol kinase does not extend to monoglycerides. ►Monoglycerides inhibit diacylglycerol kinase. ►Inhibition by monoglycerides is lower for the more specific isoform epsilon. ►These results have implications for the known role of DGKε in neuronal function.

Keywords: Abbreviations; DGK; diacylglycerol kinase; 2-AG; 2-arachidonoyl glycerol; 2-OG; 2-oleoyl glycerol; DOPC; 1,2-dioleoyl-; sn-; glycero-3-phosphocholine; DOPS; 1,2-dioleoyl-; sn; -glycero-3-[phospho-; l; -serine]; SAPA; 1-stearoyl-2-arachidonoyl phosphatidic acid; DOG; 1,2-dioleoylglycerol; SAG; 1-stearoyl-2-arachidonoylglycerol; DAG; diacylglycerol; DTT; dithiothreitol; BHT; butylated hydroxytolueneDiacylglycerol kinase; 2-arachidonoyl glycerol; 2-oleoyl glycerol; Endocannabinoid

N-cholesteryl sphingomyelin—A synthetic sphingolipid with unique membrane properties by Christian Sergelius; Shou Yamaguchi; Tetsuya Yamamoto; J. Peter Slotte; Shigeo Katsumura (pp. 1054-1062).

A sphingomyelin chimera in which the amide-linked acyl chain was replaced with cholesterol carbamate was prepared and its properties examined. The sphingomyelin/cholesterol chimera ( N-cholesterol-D-erythro-sphingomyelin) was able to form unilamellar vesicles of defined size when extruded through 200nm pore size membranes. These N-cholesteryl sphingomyelin bilayers were resistant to solubilization by Triton X-100. When N-cholesteryl sphingomyelin was added to N-palmitoyl sphingomyelin ( N-palmitoyl- d -erythro-sphingomyelin) bilayers, it increased acyl chain order as determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy. N-cholesteryl sphingomyelin was, however, not as good an inducer of membrane order compared to cholesterol on a molar basis. Differential scanning calorimetry studies further showed that the miscibility of N-cholesteryl sphingomyelin with N-palmitoyl- d -erythro-sphingomyelin bilayers was non-ideal, and the effect of N-cholesteryl sphingomyelin on the N-palmitoyl- d -erythro-sphingomyelin gel–fluid transition enthalpy differed from that seen with cholesterol. Together with N-palmitoyl- d -erythro-sphingomyelin, the N-cholesteryl sphingomyelin chimera was able to form sterol-enriched ordered domains in a fluid 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer. N-cholesteryl sphingomyelin in the absence of N-palmitoyl- d -erythro-sphingomyelin was unable to form such sterol-enriched ordered domains in a 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer. However, N-cholesteryl sphingomyelin markedly increased the affinity of cholestatrienol for N-cholesteryl sphingomyelin containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayers, suggesting that N-cholesteryl sphingomyelin was able to somehow stabilize sterol interaction in fluid bilayers. Based on our results, we conclude that N-cholesteryl sphingomyelin behaved more like a cholesterol than a sphingolipid in fluid bilayer membranes. Because N-cholesteryl sphingomyelin increased bilayer order, conferred resistance against detergent solubilization, and is not degradable by phospholipases A₂, it could constitute a good lipocomplex matrix for drug delivery vehicles.► The membrane properties of N-cholesteryl sphingomyelin was examined. ► The compound formed liquid-ordered bilayers resistant to Triton X-100. ► The compound had more cholesterol- than sphingomyelin-like properties. ► The compound could constitute a good lipocomplex matrix for drug delivery vehicles.

Keywords: Abbreviations; 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; DSC; differential scanning calorimetry; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; N; -palmitoyl-; d; -erythro; -sphingomyelin; SM; sphingomyelin; SM-C; N; -cholesteryl sphingomyelinMembrane structure; Lateral domains; Fluorescence quenching; Cholestatrienol; Membrane solubilization

Do folate-receptor targeted liposomal photosensitizers enhance photodynamic therapy selectivity? by Garcia-Diaz María García-Díaz; Santi Nonell; Ángeles Villanueva; Juan C. Stockert; Canete Magdalena Cañete; Casado Ana Casadó; Margarita Mora; Sagrista M. Lluïsa Sagristá (pp. 1063-1071).

One of the current goals in photodynamic therapy research is to enhance the selective targeting of tumor cells in order to minimize the risk and the extension of unwanted side-effects caused by normal cell damage. Special attention is given to receptor mediated delivery systems, in particular, to those targeted to folate receptor. Incorporation of a model photosensitizer (ZnTPP) into a folate-targeted liposomal formulation has been shown to lead an uptake by HeLa cells (folate receptor positive cells) 2-fold higher than the non-targeted formulation. As a result, the photocytotoxicity induced by folate-targeted liposomes was improved. This selectivity was completely inhibited with an excess of folic acid present in the cell culture media. Moreover, A549 cells (folate receptor deficient cells) have not shown variations in the liposomal incorporation. Nevertheless, the differences observed were slighter than expected. Both folate-targeted and non-targeted liposomes localize in acidic lysosomes, which confirms that the non-specific adsorptive pathway is also involved. These results are consistent with the singlet oxygen kinetics measured in living cells treated with both liposomal formulations.► Folate-targeted liposomes show enhanced ZnTPP internalization by receptor positive cells. ► Folate receptor deficient cells do not show variations in the liposomal incorporation. ► The photocytotoxicity induced by folate-targeted liposomes is improved. ► Both folate-targeted and non-targeted liposomes localize in acidic lysosomes. ►¹O₂ kinetics measured in living cells indicates a lysosome localization of ZnTPP.

Keywords: Abbreviations; BCA; bicinchoninic acid; DMEM; Dulbecco's Modified Eagle's Medium; DMSO; dimethyl sulfoxide; D-PBS; deuterated phosphate-buffered saline; FA-PEG-DSPE; 2-distearoyl-; sn; -glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (ammonium salt); FBS; fetal bovine serum; FD-DMEM; folate-deficient Dulbecco's Modified Eagle's Medium; MLV; multilamellar vesicle; MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OOPS; 1,2-dioleoyl-; sn; -glycero-3-[phospho-; L; -serine] (sodium salt); PBS; phosphate-buffered saline; PDT; photodynamic therapy; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PS; photosensitizer; SDS; sodium dodecyl sulfate; THF; tetrahydrofuran; ZnTPP; 5,10,15,20-tetraphenyl-21; H; ,23; H; -porphine zincPhotodynamic therapy; Folate receptor; Liposomes; Targeted drug delivery; Singlet oxygen

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes by Marija Raguz; Laxman Mainali; Justyna Widomska; Witold K. Subczynski (pp. 1072-1080).

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels ( n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.► The cholesterol bilayer domain (CBD) is formed within the PCD as a coexisting domain. ► The organization of cholesterol in CBD differs significantly from that in cholesterol crystals. ► The EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.

Keywords: Cholesterol bilayer domain; Lens lipid; Cholesterol; Membrane; Spin label; EPR

Membrane selectivity by W-tagging of antimicrobial peptides by Artur Schmidtchen; Lovisa Ringstad; Gopinath Kasetty; Hiroyasu Mizuno; Mark W. Rutland; Martin Malmsten (pp. 1081-1091).

A pronounced membrane selectivity is demonstrated for short, hydrophilic, and highly charged antimicrobial peptides, end-tagged with aromatic amino acid stretches. The mechanisms underlying this were investigated by a method combination of fluorescence and CD spectroscopy, ellipsometry, and Langmuir balance measurements, as well as with functional assays on cell toxicity and antimicrobial effects. End-tagging with oligotryptophan promotes peptide-induced lysis of phospholipid liposomes, as well as membrane rupture and killing of bacteria and fungi. This antimicrobial potency is accompanied by limited toxicity for human epithelial cells and low hemolysis. The functional selectivity displayed correlates to a pronounced selectivity of such peptides for anionic lipid membranes, combined with a markedly reduced membrane activity in the presence of cholesterol. As exemplified for GRR10W4N (GRRPRPRPRPWWWW-NH₂), potent liposome rupture occurs for anionic lipid systems (dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) and Escherichia coli lipid extract) while that of zwitterionic dioleoylphosphatidylcholine (DOPC)/cholesterol is largely absent under the conditions investigated. This pronounced membrane selectivity is due to both a lower peptide binding to the zwitterionic membranes ( z≈−8–10mV) than to the anionic ones ( z≈−35–40mV), and a lower degree of membrane incorporation in the zwitterionic membranes, particularly in the presence of cholesterol. Replacing cholesterol with ergosterol, thus mimicking fungal membranes, results in an increased sensitivity for peptide-induced lysis, in analogy to the antifungal properties of such peptides. Finally, the generality of the high membrane selectivity for other peptides of this type is demonstrated.► Membrane selectivity is shown for W/F-tagged short and highly charged peptides. ► These display broad-spectrum antimicrobial effect, but low toxicity to human cells. ► Parallel selectivity for anionic and cholesterol-void lipid membranes. ► Replacing cholesterol with ergosterol yields increased peptide-induced lysis. ► The generality of the membrane selectivity for related peptides is demonstrated.

Keywords: AMP; Antimicrobial peptide; Ellipsometry; Liposome; Membrane; Selectivity

Lipid-binding surfaces of membrane proteins: Evidence from evolutionary and structural analysis by Larisa Adamian; Hammad Naveed; Jie Liang (pp. 1092-1102).

Membrane proteins function in the diverse environment of the lipid bilayer. Experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface. These lipid molecules often act as co-factors and play important functional roles. In this study, we have assessed the evolutionary selection pressure experienced at lipid-binding sites in a set of α-helical and β-barrel membrane proteins using posterior probability analysis of the ratio of synonymous vs. nonsynonymous substitutions (ω-ratio). We have also carried out a geometric analysis of the membrane protein structures to identify residues in close contact with co-crystallized lipids. We found that residues forming cholesterol-binding sites in both β₂-adrenergic receptor and Na⁺–K⁺-ATPase exhibit strong conservation, which can be characterized by an expanded cholesterol consensus motif for GPCRs. Our results suggest the functional importance of aromatic stacking interactions and interhelical hydrogen bonds in facilitating protein–cholesterol interactions, which is now reflected in the expanded motif. We also find that residues forming the cardiolipin-binding site in formate dehydrogenase-N γ-subunit and the phosphatidylglycerol binding site in KcsA are under strong purifying selection pressure. Although the lipopolysaccharide (LPS)-binding site in ferric hydroxamate uptake receptor (FhuA) is only weakly conserved, we show using a statistical mechanical model that LPS binds to the least stable FhuA β-strand and protects it from the bulk lipid. Our results suggest that specific lipid binding may be a general mechanism employed by β-barrel membrane proteins to stabilize weakly stable regions. Overall, we find that the residues forming specific lipid binding sites on the surfaces of membrane proteins often experience strong purifying selection pressure.► Binding sites for functionally important lipids are often under strong purifying selection. ► Expanded cholesterol binding motif with interhelical H-bond (S,G)–(W,Y). ► Specific lipid binding stabilizes weakly stable regions.

Keywords: Lipid binding; ω-ratio; Cholesterol binding motif; Lipopolysaccharide

Patch clamp analysis of the respiratory chain in Bacillus subtilis by Koji Nakamura; Satoshi Ikeda; Taira Matsuo; Aiko Hirata; Masaya Takehara; Tetsuo Hiyama; Fujio Kawamura; Iwao Kusaka; Tomofusa Tsuchiya; Teruo Kuroda; Isamu Yabe (pp. 1103-1107).

Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H⁺ translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline- N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H⁺ translocation mechanism.Display Omitted► Bacillus subtilis cells are enlarged as large as 10–20 μm in diameter. ► Giant protoplasts contain a vacuole-like structure, named provacuole. ► Provacuole is like an everted membrane vesicle. ► Patch clamp technique is applicable to provacuole. ► NADH-induced current was observed in provacuole.

Keywords: Abbreviations; GP; giant protoplast; GV; giant provacuole; EtBr; ethidium bromide; HQNO; 2-heptyl-4-hydroxyquinoline-; N; -oxidePatch clamp; Respiratory chain; Bacillus subtilis; Giant protoplast

Pun1p is a metal ion-inducible, calcineurin/Crz1p-regulated plasma membrane protein required for cell wall integrity by Dagmar Hosiner; Gerhard Sponder; Anton Graschopf; Siegfried Reipert; Rudolf J. Schweyen; Schuller Christoph Schüller; Markus Aleschko (pp. 1108-1119).

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 ( YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments. ► PUN1 is induced upon metal ion stress. ► Deletion of PUN1 causes metal ion sensitivity and a yeast cell wall defect. ► Under metal ion stress PUN1 is regulated by the calcineurin/Crz1p pathway. ► Pun1p forms higher-order protein complexes.

Keywords: Abbreviations; MCC; membrane compartment occupied by Can1; ICP-MS; inductively coupled plasma mass spectrometry; DIC; differential interference contrast; TEM; transmission electron microscope; HOG; high osmolarity glycerol; o-PDM; o-phenylenedimaleimide; D; digitonin; TX; Triton X-100; DM; n-Dodecyl-β-; d; -maltoside; Co-IP; Co-ImmunoprecipitationCell wall; Plasma membrane; Metal ion stress; Calcineurin/Crz1p; Oligomerization

The pore properties of human nociceptor channel TRPA1 evaluated in single channel recordings by Y.V. Bobkov; E.A. Corey; B.W. Ache (pp. 1120-1128).

TRPA channels detect stimuli of different sensory modalities, including a broad spectrum of chemosensory stimuli, noxious stimuli associated with tissue damage and inflammation, mechanical stimuli, and thermal stimuli. Despite a growing understanding of potential modulators, agonists, and antagonists for these channels, the exact mechanisms of channel regulation and activation remain mostly unknown or controversial and widely debated. Relatively little is also known about the basic biophysical parameters of both native and heterologously expressed TRPA channels. Here we use conventional single channel inside-out and outside-out patch recording from the human TRPA1 channel transiently expressed in human embryonic kidney 293T cells to characterize the selectivity of the channel for inorganic mono-/divalent and organic monovalent cations in the presence of allylisothiocyanate (AITC). We show the relative permeability of the hTRPA1 channel to inorganic cations to be:Ca2+5.1>Ba2+3.5>Mg2+2.8>NH4+1.5>Li+1.2>Na+1.0≥K+0.98≥Rb+0.98>Cs+0.95;and to organic cations:Na⁺(1.0)≥dimethylamine (0.99)>trimethylamine (0.7)>tetramethylammonium (0.4)> N-methyl-d-glucamine (0.1). Activation of the hTRPA1 channels by AITC appears to recruit the channels to a conformational state with an increased permeability to large organic cations. The pore of the channels in this state can be characterized as dilated by approximately 1–2.5 Å. These findings provide important insight into the basic fundamental properties and function of TRPA1 channels in general and human TRPA1 channel in particular.► Human TRPA1 channel weakly discriminates among both mono- and divalent cations. ► P_X⁺/ P_Na⁺s are Li⁺ (1.26)≥K⁺ (0.98)≥Rb⁺ (0.98)>Cs⁺ (0.95). ► P_Y²⁺/ P_Na⁺s are Ca²⁺ (5.1)>Ba²⁺ (3.5)>Mg²⁺ (2.8). ► Some ligands recruit the hTRPA1 channel to a state permeable to large organic cations. ► The pore of the channel in this state is dilated by approximately 1–2.5 Å

Keywords: Human TRPA channel; Ionic permeability; Selectivity; Channel pore diameter

Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers by Ofelia Maniti; Mouhedine Cheniour; Marie-France Lecompte; Olivier Marcillat; René Buchet; Christian Vial; Thierry Granjon (pp. 1129-1139).

It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein–cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested.►Formation of mtCK-cardiolipin clusters depends on CL acyl chain composition. ►mtCK induces clustering of unsaturated egg yolk PG, but not of saturated DMPG. ►mtCK reduces membrane fluidity both for TMCL and bovine heart CL-containing liposomes. ►Protein segments penetrate both saturated TMCL and DMPG and unsaturated bovine heart CL and PG monolayers. ►Protein domain movements occur after binding to saturated and unsaturated cardiolipins or phosphatidylglycerols, with the highest amplitude observed for bovine heart CL.

Keywords: Mitochondrial creatine kinase; Cardiolipin; Lipid domain; Acyl chain composition; Langmuir monolayer; Brewster angle microscopy

Vulnerability of the cysteine-less proton-coupled folate transporter (PCFT-SLC46A1) to mutational stress associated with the substituted cysteine accessibility method by Rongbao Zhao; Daniel Sanghoon Shin; I. David Goldman (pp. 1140-1145).

The proton-coupled transporter (PCFT) mediates intestinal folate absorption and folate transport from blood across the choroid plexus. The membrane topology of PCFT has been defined using the substituted cysteine accessibility method; an intramolecular disulfide bond between the Cys 66 and 298 residues, in the first and fourth extracellular loops, respectively, is present but not essential for function. The current report describes Lys 422 mutations (K422C, K422E) that have no effect on transport activity when introduced into wild-type PCFT but result in a marked loss of activity when introduced into a Cys-less PCFT which is otherwise near-fully functional. The loss of activity of both mutant PCFTs was shown to be due to impaired protein stability and expression. Additional studies were conducted with the K422C mutation in Cys-less PCFT. The impact of re-introduction of one, two, three or five, Cys residues was assessed. While there were some differences in the impact of the different Cys residues re-introduced, restoration was attributed more to a cumulative effect rather than the specific role of individual Cys residues. Preservation of the Cys66–Cys298 intramolecular disulfide bond was not required for stability of the K422C protein. These observations are relevant to studies with Cys-less transporters utilized for the characterization of proteins with the substituted cysteine accessibility method and indicate that functional defects detected in a Cys-less protein, when the tertiary structure of the molecule is stressed, are not necessarily relevant to the wild-type protein.► The highly conserved Lys 422 is fully replaceable in wild-type PCFT. ► K422C and K422E mutants are inactive in the functional Cys-less PCFT. ► The K422C in Cys-less PCFT loses activity due to decreased protein expression. ► Additions of Cys residues cumulatively restore K422C PCFT expression and activity.

Keywords: Abbreviations; CL; Cysteine-less; HA; hemagglutinin; MTX; methotrexate; PCFT; the proton-coupled folate transporter; HFM; hereditary folate malabsorption; SCAM; substituted cysteine accessibility methodProton-coupled folate transporter; Hereditary folate malabsorption; Substituted cysteine accessibility method; Folate

Fatty acid composition modulates sensitivity of Legionella pneumophila to warnericin RK, an antimicrobial peptide by Julien Verdon; Jérome Labanowski; Tobias Sahr; Thierry Ferreira; Christian Lacombe; Carmen Buchrieser; Jean-Marc Berjeaud; Hechard Yann Héchard (pp. 1146-1153).

Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires’ disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti- Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid chain length in membrane were correlated with the increase in resistance to warnericin RK. Therefore, fatty acid profile seems to play a critical role in the sensitivity of L. pneumophila to warnericin RK.► Growth phase modulates sensitivity to warnericin RK and fatty acid composition. ► Selection of an adapted strain, resistant to warnericin RK by selection pressure experiments. ► The adapted strain is more resistant and displays higher amount of BCFA. ► Two fatty acid associated genes were repressed in the adapted strain.

Keywords: Abbreviations; AMPs; antimicrobial peptides; BCFA; branched-chain fatty acids; FAMEs; fatty acid methyl esters; PI; ₅₀; permeabilization index 50Adaptation; Branched-chain fatty acids; Membrane fluidity; Microarray; Resistance

Oligomerization of daptomycin on membranes by Jawad K. Muraih; Andre Pearson; Jared Silverman; Michael Palmer (pp. 1154-1160).

Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by membrane depolarization. While it has long been assumed that the mode of action of daptomycin involves the formation of membrane-associated oligomers, this has so far not been experimentally demonstrated. We here use FRET between native daptomycin and an NBD-labeled daptomycin derivative to show that such oligomerization indeed occurs. The oligomers are observed in the presence of calcium ions on membrane vesicles isolated from Bacillus subtilis, as well as on model membranes containing the negatively charged phospholipid phosphatidylglycerol. In contrast, oligomerization does not occur on membranes containing phosphatidylcholine only, nor in solution at micromolar daptomycin concentrations. The requirements for oligomerization of daptomycin resemble those previously reported for antibacterial activity, suggesting that oligomerization is necessary for the activity.► Daptomycin is shown to form oligomers on liposome membranes. ► Oligomerization depends on presence of phosphatidylglycerol; phosphatidylcholine membranes bind daptomycin but do not induce oligomerization. ► Lipid requirements for oligomerization correlate with those for antimicrobial activity.

The amyloidogenic SEVI precursor, PAP248-286, is highly unfolded in solution despite an underlying helical tendency by Jeffrey R. Brender; Ravi Prakash Reddy Nanga; Nataliya Popovych; Ronald Soong; Peter M. Macdonald; Ayyalusamy Ramamoorthy (pp. 1161-1169).

Amyloid fibers in human semen known as SEVI (semen-derived enhancer of viral infection) dramatically increase the infectivity of HIV and other enveloped viruses, which appears to be linked to the promotion of bridging interactions and the neutralization of electrostatic repulsion between the host and the viral cell membranes. The SEVI precursor PAP_248–286 is mostly disordered when bound to detergent micelles, in contrast to the highly α-helical structures found for most amyloid proteins. To determine the origin of this difference, the structures of PAP_248–286 were solved in aqueous solution and with 30% and 50% trifluoroethanol. In solution, pulsed field gradient (PFG)-NMR and¹H-¹H NOESY experiments indicate that PAP_248–286 is unfolded to an unusual degree for an amyloidogenic peptide but adopts significantly helical structures in TFE solutions. The clear differences between the structures of PAP_248–286 in TFE and SDS indicate electrostatic interactions play a large role in the folding of the peptide, consistent with the slight degree of penetration of PAP_248–286 into the hydrophobic core of the micelle. This is another noticeable difference between PAP_248–286 and other amyloid peptides, which generally show penetration into at least the headgroup region of the bilayer, and may explain some of the unusual properties of SEVI.Display Omitted► PAP_248–286 folds into a disordered structure upon binding to SDS micelles. ► PAP_248–286 is significantly unfolded in solution. ► PAP_248–286 adopts helical structures in TFE solutions. ► Disordered structure of PAP_248–286 in SDS results from electrostatic interactions. ► Unusual degree of disorder of PAP_248–286 may account for its unusual properties.

Keywords: Amyloid; SEVI; Natively disordered; Structure; NMR

Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β₂-adrenergic receptor by Makoto Arakawa; Raja Chakraborty; Jasbir Upadhyaya; Markus Eilers; Philip J. Reeves; Steven O. Smith; Prashen Chelikani (pp. 1170-1178).

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix–helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2–H4 in the β₂-adrenergic receptor (β₂-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315^7.42 and Ser319^7.46, on H7 for structure-function analysis. Replacing Ser319^7.46 with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15–20% agonist-independent activity. Replacement of Ser319^7.46 with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315^7.42 and Ser319^7.46 are stabilizing β₂-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315^7.42 with Trp286^6.48 and hydrogen bonding interactions of Ser319^7.46 with amino acids on H1–H2–H7 and with structural water.► Gly315^7.42 and Ser319^7.46 stabilize β₂-AR in an inactive conformation. ► Occluded surface calculations show Gly315^7.42 does not form a water binding site. ► Gly315^7.42 acts as a “molecular notch” to stabilize Trp286^6.48 in an inactive position. ► Ser319^7.46 mediates hydrogen bonding interactions between Trp286^6.48 and Asn322^7.49.

Keywords: Abbreviations; β; ₂; -AR; β; ₂; -adrenergic receptor; B; _max; Binding maximum of the ligand for the receptor; COS-1; Monkey kidney cells; GPCRs; G protein-coupled receptors; HEK293; human embryonic kidney cells; [; ³; H] DHA; tritium labeled dihydroalprenolol; K; _d; equilibrium dissociation constant of the ligandG-protein coupled receptors (GPCRs); Helix packing; Beta2-adrenergic receptor; GPCR activation; Site-directed mutagenesis.

N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol by Bjorkbom Anders Björkbom; Rog Tomasz Róg; Kankaanpaa Pasi Kankaanpää; Daniel Lindroos; Karol Kaszuba; Mayuko Kurita; Shou Yamaguchi; Tetsuya Yamamoto; Shishir Jaikishan; Lassi Paavolainen; Paivarinne Joacim Päivärinne; Thomas K.M. Nyholm; Shigeo Katsumura; Ilpo Vattulainen; J. Peter Slotte (pp. 1179-1186).

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability ( T_m was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.► Sphingomyelin analogs were prepared which had methylations on the 2NH- or the 3OH-function, or both. ► The bilayer properties of the SM analogs were determined experimentally and by molecular dynamics simulations. ► Methylations also markedly attenuated interactions with sterols. ► MD simulations further showed that cholesterol's depth and tilt angle in the bilayer were affected. ► We conclude that interfacial properties markedly influence molecular and domain-forming properties of SMs.

Keywords: Hydrogen bonding; Acyl chain order; Molecular dynamics simulation; Sterol partitioning; Lateral domains

Temperature–pressure phase diagram of a heterogeneous anionic model biomembrane system: Results from a combined calorimetry, spectroscopy and microscopy study by Shobhna Kapoor; Werkmuller Alexander Werkmüller; Christian Denter; Yong Zhai; Jonas Markgraf; Katrin Weise; Norbert Opitz; Roland Winter (pp. 1187-1195).

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature–pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10–20 mol% PG still leads to liquid-ordered (l_o)-liquid-disordered (l_d) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p, T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature–pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.► The T, p-phase diagram of an anionic model raft mixture has been determined. ► A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered. ► Biological studies under extreme environmental conditions are feasible, now.

Keywords: High pressure; Lipid bilayers; Model raft mixture; FTIR; DSC; Fluorescence microscopy; Fluorescence spectroscopy; AFM

Visualization of ceramide channels by transmission electron microscopy by Soumya Samanta; Johnny Stiban; Timothy K. Maugel; Marco Colombini (pp. 1196-1201).

Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.► Ceramide self-assembles in single wall liposomes to form large stable channels. ► The channels were visualized by negative staining and electron microscopy (EM). ► Channels are cylindrical in nature. ► Channel diameter is not strictly limited by the energetics of channel curvature. ► The channel size distribution observed by EM matches with electrophysiological data.

Keywords: Channel; Negative stain; Pore; Ceramide; Sphingolipid; Apoptosis

Identification of a novel endoplasmic reticulum export motif within the eighth α-helical domain (α-H8) of the human prostacyclin receptor by Peter D. Donnellan; Cisca C. Kimbembe; Helen M. Reid; B. Therese Kinsella (pp. 1202-1218).

The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val²⁹⁹–Val³⁰⁷ within the eighth helical domain (α-H8) adjacent to the palmitoylated residues at Cys³⁰⁸–Cys³¹¹. However, the factors determining the anterograde transport of the newly synthesised hIP from the endoplasmic reticulum (ER) to the plasma membrane (PM) have not been identified. The aim of the current study was to identify the major ER export motif(s) within the hIP initially by investigating the role of Lys residues in its maturation and processing. Through site-directed and Ala-scanning mutational studies in combination with analyses of protein expression and maturation, functional analyses of ligand binding, agonist-induced intracellular signalling and confocal image analyses, it was determined that Lys²⁹⁷, Arg³⁰² and Lys³⁰⁴ located within α-H8 represent the critical determinants of a novel ER export motif of the hIP. Furthermore, while substitution of those critical residues significantly impaired maturation and processing of the hIP, replacement of the positively charged Lys with Arg residues, and vice versa, was functionally permissible. Hence, this study has identified a novel 8 residue ER export motif within the functionally important α-H8 of the hIP. This ER export motif, defined by “K/R(X)₄K/R(X)K/R,” has a strict requirement for positively charged, basic Lys/Arg residues at the 1st, 6th and 8th positions and appears to be evolutionarily conserved within IP sequences from mouse to man.► Identification of a novel ER export motif in the human prostacyclin receptor (hIP). ► Lys & Arg in the α-helical 8 domain of the hIP are critical for the ER export motif. ► The ER export motif may be defined as K/R(X)₄K/R(X)K/R, where X is any amino acid. ► The ER export motif is evolutionary conserved in the IPs from mouse to man.

Keywords: Abbreviations; α-H8; α-helix 8; AC; adenylyl cyclase; [Ca; ²⁺; ]; _i; intracellular calcium; C-tail; carboxyl-terminal tail; COPII vesicles; coat protein-II vesicles; ER; endoplasmic reticulum; ERAD; ER-associated degradation; EC; extracellular; GPCR; G protein-coupled receptor; HA; hemagglutinin; HEK; human embryonic kidney; hIP; human IP; IC; intracellular; IP; PGI; ₂; receptor; PG; prostaglandin; PM; plasma membrane; RBD; Rab11 binding domain; TM; transmembraneGPCR; Prostacyclin receptor; Alpha helix 8; Trafficking; Endoplasmic reticulum; Export

Membrane interaction of segment H1 (NS4B_H1) from hepatitis C virus non-structural protein 4B by M. Francisca Palomares-Jerez; Villalain José Villalaín (pp. 1219-1229).

NS4B protein from hepatitis C virus (HCV) is a highly hydrophobic protein inducing a rearrangement of endoplasmic reticulum membranes responsible of the HCV replication process. Different helical elements have been found in the N- and C- terminal domains of the protein, which seem to be responsible for many key aspects of the viral replication process. In this work we have carried out a study of the binding and interaction with model biomembranes of peptide NS4B_H1, patterned after segment H1, one of these C-terminal previously identified segments. We show that NS4B_H1 partitions into phospholipid membranes; its membrane activity is modulated by lipid composition, interacting preferentially with negatively charged phospholipids as well as with sphingomyelin. Furthermore, the change in its sequence prevents the resulting peptide from interacting with the membrane. These data would support its role in the interaction of NS4B with the membrane and suggest that the region where this peptide resides could be involved in the membrane alteration which must occur in the HCV replication and/or assembly process.► We have studied the binding and interaction of peptide NS4BH1 from HCV NS4B with model membranes. ► NS4BH1 partitions into and interacts with membrane phospholipid membranes. ► NS4BH1 sequence is fundamental for its activity. ► NS4BH1 structure is modulated by membrane binding. ► NS4BH1 region might be implicated in HCV replication process Página

Keywords: Abbreviations; 16-NS; 16-Doxyl-stearic acid; 5-NS; 5-Doxyl-stearic acid; BPI; Bovine brain L-α-phosphatidylinositol; BPS; Bovine brain L-α-phosphatidylserine; CF; 5-Carboxyfluorescein; CHOL; Cholesterol; CL; Bovine heart cardiolipin; DMPA; 1,2-Dimyristoyl-; sn; -glycero-3-phosphatidic acid; DMPC; 1,2-Dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DMPG; 1,2-Dimyristoyl-; sn; -glycero-3-[phospho-rac-glycerol]; DMPS; 1,2-Dimyristoyl-; sn; -glycero-3-phosphatidylserine; DPH; 1,6-Diphenyl-1,3,5-hexatriene; DSC; Differential scanning calorimetry; EPA; Egg L-α-phosphatidic acid; EPC; Egg L-α-phosphatidylcholine; EPG; Egg L-α-phosphatidylglycerol; ER; Endoplasmic reticulum; ESM; Egg sphingomyelin; HCV; Hepatitis C virus; IR; Infrared spectroscopy; LUV; Large unilamellar vesicles; MLV; Multilamellar vesicles; NS; Non-structural protein; PSM; N-palmitoyl-D-erythro-sphingosylphosphorylcholine; SUV; Small unilamellar vesicles; TFE; Trifluoroethanol; T; _m; Temperature of the gel-to-liquid crystalline phase transition; TMA-DPH; 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TPE; Egg trans-esterified L-α-phosphatidylethanolamineHCV replication; HCV; Lipid–peptide interaction; Membranous web; NS4B H1

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BBA - Biomembranes (v.1808, #4)