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BBA - Biomembranes (v.1712, #2)

Editorial Board (pp. ii).

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy by Simon Scheuring; Daniel L�vy; Jean-Louis Rigaud (pp. 109-127).

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll–protein complexes composing the photosynthetic apparatus in different species of purple bacteria.

Keywords: Abbreviations; AFM; Atomic Force Microscopy; EM; Electron Microscopy; LH; light-harvesting; RC; reaction centre; 4Hcyt; tetraheme cytochrome; cyt; bc1; Cytochrome; bc1; complex; PSU; photosynthetic unit; Rb.; Rhodobacter; Rsp.; Rhodospirillum; Blc; .; Blastochloris; Rvi.; Rubrivivax; Rps.; RhodopseudomonasAFM; Photosynthesis; Purple photosynthetic bacteria; Light harvesting complexes; Reaction centre; Core complex; Photosynthetic unit; Blastochloris viridis; Rhodospirillum photometricum; Rhodobacter blasticus; Rhodobacter sphaeroides

Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes by Vasyl V. Shynkar; Andrey S. Klymchenko; Guy Duportail; Alexander P. Demchenko; Yves M�ly (pp. 128-136).

The dipole potential ( Ψ_d) constitutes a large and functionally important part of the electrostatic potential of cell plasma membranes. However, its direct measurement is not possible. Herein, new 3-hydroxyflavone fluorescent probes were developed that respond strongly to Ψ_d changes by a variation of the intensity ratio of their two well-separated fluorescence bands. Using fluorescence spectroscopy with cell suspensions and confocal microscopy with adherent cells, we showed, for the first time, two-color fluorescence ratiometric measurement and visualization of Ψ_d in cell plasma membranes. Using this new tool, a heterogeneous distribution of this potential within the membrane was evidenced.

Keywords: 3-Hydroxyflavone derivative; Cell plasma membrane; Confocal fluorescence microscopy; Dipole potential; Fluorescence probe; Fluorescence spectroscopy

Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover by Bao-Zhu Yu; Tatyana Polenova; Mahendra Kumar Jain; Otto G. Berg (pp. 137-151).

During the steady state reaction progress in the scooting mode with highly processive turnover, Bacillus cereus sphingomyelinase (SMase) remains tightly bound to sphingomyelin (SM) vesicles (Yu et al., Biochim. Biophys. Acta 1583, 121–131, 2002). In this paper, we analyze the kinetics of SMase-catalyzed hydrolysis of SM dispersed in diheptanoylphosphatidyl-choline (DC₇PC) micelles. Results show that the resulting decrease in the turnover processivity induces the stationary phase in the reaction progress. The exchange of the bound enzyme (E*) between the vesicle during such reaction progress is mediated via the premicellar complexes (E_i^#) of SMase with DC₇PC. Biophysical studies indicate that in E_i^# monodisperse DC₇PC is bound to the interface binding surface (i-face) of SMase that is also involved in its binding to micelles or vesicles. In the presence of magnesium, required for the catalytic turnover, three different complexes of SMase with monodisperse DC₇PC (E_i^# with i=1, 2, 3) are sequentially formed with Hill coefficients of 3, 4 and 8, respectively. As a result, during the stationary phase reaction progress, the initial rate is linear for an extended period and all the substrate in the reaction mixture is hydrolyzed at the end of the reaction progress. At low mole fraction ( X) of total added SM, exchange is rapid and the processive turnover is limited by the steps of the interfacial turnover cycle without becoming microscopically limited by local substrate depletion or enzyme exchange. At high X, less DC₇PC will be monodisperse, E_i^# does not form and the turnover becomes limited by slow enzyme exchange. Transferred NOESY enhancement results show that monomeric DC₇PC in solution is in a rapid exchange with that bound to E_i^# at a rate comparable to that in micelles. Significance of the exchange and equilibrium properties of the E_i^# complexes for the interpretation of the stationary phase reaction progress is discussed.

Keywords: Abbreviations; DC; ₇; PC; Diheptanoylphosphatidylcholine; HDNS; N-dansyl-hexadecyl-1-phosphoethanolamine; i-face; the interface binding surface of an interfacial enzyme; ITC; isothermal calorimetry; RET; fluorescence resonance energy transfer; SM; sphingomyelin; SMase; sphingomyelinase from; Bacillus cereus; TMA-DPH; trimethylammonium-diphenylhexatrieneSphingomyelinase; Bacillus cereus; Interfacial catalytic turnover; Pseudo-scooting mode; Premicellar complex; Transferred NOESY

Giant liposomes in physiological buffer using electroformation in a flow chamber by Daniel J. Estes; Michael Mayer (pp. 152-160).

We describe a method to obtain giant liposomes (diameter 10–100 μm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.

Keywords: Giant liposome; Electroformation; Physiological buffer; Membrane-binding assay; Annexin V

Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides by James R. Maiolo; Marc Ferrer; Elizabeth A. Ottinger (pp. 161-172).

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R₇, and R₇W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.

Keywords: Abbreviations; CPP; cell penetrating peptide; DIEA; diisopropylethylamine; DMEM; Dubelcco’s Modified Eagle Medium; DMF; dimethylformamide; DMPC; dimyristoyl phosphatidylcholine; DMPG; dimyristoyl phosphidylglycerol; DMSO; dimethylsulfoxide; FBS; fetal bovine serum; Fl; 5-carboxyfluorescein; Fmoc; N-(9-fluorenyl)methoxycarbonyl; FP; fluorescence polarization; HBTU; 2-(1H-benzotriazole -1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt; 1-hydroxybenzotriazole; NMP; N; -methylpyrrolidinone; pAntp; antennapedia; PBS; phosphate-buffered saline; POPC; palmitoyl-oleoyl phosphatidylcholine; POPG; palmitoyl-oleoyl phosphatidylglycerol; SUV; small-unilamellar vesicles; TFA; trifluoroacetic acidConjugated cell-penetrating peptide; Arginine-rich peptide; Cellular distribution; Quantitative fluorescence microscopy; Endocytosis; Lipid binding

Membrane topology of loop 13–14 of the Na⁺/glucose cotransporter (SGLT1): A SCAM and fluorescent labelling study by Dominique G. Gagnon; Andrea Holt; Francis Bourgeois; Bernadette Wallendorff; Michael J. Coady; Jean-Yves Lapointe (pp. 173-184).

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na⁺/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES⁽⁻⁾ and MTSET⁽⁺⁾ had no effect on either cotransport or Na⁺ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K_i^Pz and K_m^αMG for wtSGLT1 and for cysteine mutants at positions 588, 605–608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K_i^Pz values than wtSGLT1 with minimal changes in K_m^αMG, the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.

Keywords: Abbreviations; SGLT1; high affinity Na; ⁺; /glucose cotransporter; hSGLT1; human isoform of SGLT1; rSGLT1; rabbit isoform of SGLT1; wtSGLT1; wild-type SGLT1; SCAM; substituted cysteine accessibility method; MTS; methanethiosulfonate; MTSES; sodium (2-sulfonatoethyl)methanethiosulfonate; MTSET; [2-(Trimethylammonium) ethyl]-methanethiosulfonate bromide; MTSEA; 2-aminoethyl methanethiosulfonate hydrobromide; TMR5(6)M; tetramethylrhodamine-5(or 6)-maleimide; TMS; transmembrane segment; Pz; phlorizin; αMG; α−Methyl-glucose; V; _m; membrane potential; I; _cotr; αMG cotransport current; I; _leak; Na; ⁺; leak current; AA; amino acid; K; _m; ^αMG; apparent affinity for αMG; K; _i; ^Pz; inhibition constant of Pz; K; _m; ^Na+; apparent affinity for Na; ⁺; VSVG; vesicular stomatitis virus G proteinNa; ⁺; /glucose cotransporter; Electrophysiology; SCAM; Fluorescence; Topology

Characterization of the ion transport activity of the budding yeast Na⁺/H⁺ antiporter, Nha1p, using isolated secretory vesicles by Ryuichi Ohgaki; Norihiro Nakamura; Keiji Mitsui; Hiroshi Kanazawa (pp. 185-196).

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.

Keywords: Abbreviations; NHA; Na; ⁺; /H; ⁺; antiporter; NHE; Na; ⁺; /H; ⁺; exchanger; SDS PAGE; SDS polyacrylamide gel electrophoresis; ORF; Open Reading Frame; ACMA; 9-Amino-6-Chloro-2-Methoxyacridine; EGFP; Enhanced Green Fluorescent Protein; pmf; proton motive force; CCCP; Carbonyl Cyanide; m; -ChlorophenylhydrazoneYeast Na; ⁺; /H; ⁺; antiporter; Ion selectivity; Electrogenicity; Yeast secretory vesicle

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43–58) by Michael E. Herbig; Ursina Fromm; Jeannine Leuenberger; Ulrike Krauss; Annette G. Beck-Sickinger; Hans P. Merkle (pp. 197-211).

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9–32) and its derivatives, D values raised from about 100–200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide–phospholipid interactions, especially when negatively charged lipids were present.

Keywords: Abbreviations; CPP; cell penetrating peptide; hCT; human calcitonin; pVEC; vascular endothelial cadherin-derived CPP; pAntp; penetratin, Antennapedia homeodomain-derived CPP; POPC; 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-phosphatidylglycerol; DPC; dodecyl phosphocholine; Br-PC; 1-palmitoyl-2-stearoyl-(11,12-dibromo)-; sn; -glycero-3-phosphocholine; NBD-PE; N-(7-nitrobenzofurazan-4-yl)-1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine; 5-DSA; 5-doxylstearic acid; DPH; 1,6-diphenyl-1,3,5-hexatriene; TFA; trifluoroacetic acid; PBS; phosphate-buffered saline; RP-HPLC; reversed phase HPLC; LUV; large unilamellar vesicles; SUV; small unilamellar vesicles; MLV; multilamellar vesicles; DLS; dynamic light scattering; K; _SV; Stern–Volmer constantCell penetrating peptides; Fluorescence spectroscopy; Liposome–buffer partitioning; Lipid bilayer models; Phospholipid vesicles; Peptide–lipid interactions

Chondrocyte transport and concentration of ascorbic acid is mediated by SVCT2 by Amy L. McNulty; Thomas P. Vail; Virginia B. Kraus (pp. 212-221).

Collagen II is the major protein component of articular cartilage and forms the collagen fibril network, which provides the tensile strength of cartilage. Collagen II synthesis is enhanced by ascorbic acid (vitamin C) at both a transcriptional and post-transcriptional level. While the importance of ascorbic acid in the synthesis of collagen has been established, the mechanism by which this essential nutrient is transported into chondrocytes has not been investigated previously. We have characterized the transport of the reduced form of ascorbic acid in passaged primary human chondrocytes to discern the physiologically relevant pathways of ascorbic acid transport in cartilage. We have found that chondrocytes are robust concentrators of ascorbic acid, capable of transporting the reduced form, and concentrating total ascorbic acid, in the reduced form and its metabolites, 960-fold over the concentration in the extracellular milieu. Chondrocyte transport of ascorbic acid was sodium and temperature dependent, stereoselective for the L-forms, and inhibited by the anion transport inhibitor, sulfinpyrazone. Chondrocytes preferentially expressed the full-length and functional isoform of sodium-dependent vitamin C transporter 2 (SVCT2). When this transcript was suppressed with sequence-specific siRNAs, the active transport component of ascorbic acid was abolished. Thus, we provide the first evidence that SVCT2 mediates the secondary active and concentrative transport of ascorbic acid in human chondrocytes.

Keywords: Ascorbic acid; Sodium-dependent vitamin C transporter; Dehydroascorbate; Glucose transporter; Chondrocyte; Vitamin C

Erratum to “Intestinal bicarbonate secretion by marine teleost fish—Why and how?�? [Biochim. Biophys. Acta, Biomembr. 1566 (2002) 182–193] by Rod W. Wilson; Jonathan M. Wilson; Martin Grosell (pp. 222-222).

Cumulative contents (pp. 223-224).

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BBA - Biomembranes (v.1712, #2)