Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na⁺/H⁺ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na⁺/H⁺ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na⁺/H⁺ exchange function (NH₄-prepulse acid load sustained in Na⁺-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na⁺ removal revealed that only NHE3-CFP translocated when medium Na⁺ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na⁺-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts

Sulfogalactosylglycerolipid (SGG) is found in detergent-resistant lipid raft fractions isolated from sperm plasma membranes and has been shown to be important in sperm-egg adhesion. In order to provide more direct evidence for the association of sulfoglycolipids with lipid raft domains, we have examined the distribution of two sulfoglycolipids in supported membranes prepared from artificial lipid mixtures and cellular lipid extracts. Atomic force microscopy has been used to visualize the localization of SGG and sulfogalactosylceramide (SGC) in liquid-ordered domains in supported bilayers of ternary lipid mixtures comprised of dipalmitoylphosphatidylcholine, cholesterol and palmitoyldocosahexaenoylphosphatidylcholine. The localization of SGC/SGG in the liquid-ordered raft domains is demonstrated by changes in bilayer morphology in the presence of sulfoglycolipid, by selective antibody labeling of the domains with anti-SGC/SGG and by the effects of the cholesterol-sequestering agent, methyl-β-cyclodextrin, on the supported membranes. In addition, we use a combination of atomic force microscopy and immunofluorescence to show that supported bilayers made from lipids extracted from sperm anterior head plasma membranes (APM) and isolated APM vesicles exhibit small SGG-rich domains that are similar to those observed in bilayers of artificial lipid mixtures. The possible implications of these results for the involvement of SGG-rich lipid rafts in modulating sperm-egg interactions in vivo and the utility of model membranes for studying the behavior of lipid rafts are discussed.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na⁺ dependent [³H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K⁺. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [³H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na⁺and internal K⁺. The transporter showed low if there is any tolerance towards the substitution of Na⁺ or K⁺ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na⁺ concentration cooperativity index close to 1 was derived, indicating that 1 Na⁺ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na⁺ transport. Optimal rate of 0.1 mM [³H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [³H]glutamine (and [³H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl₂, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na⁺ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.     

Inhibition of plasma membrane Ca-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca-occluded state

The bidentate complex of ATP with Cr³⁺, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca²⁺ and Na⁺, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca²⁺-ATPase. The complex inhibited with similar efficiency the Ca²⁺-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T_1/2 = 30 min at 37 °C) with a K_i = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg²⁺ but unaltered when Ca²⁺ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca²⁺ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La³⁺ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca²⁺ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca²⁺ promoted by the plasma membrane Ca²⁺-ATPase goes through an enzymatic phospho-intermediate that maintains Ca²⁺ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Caenorhabditis elegans galectins LEC-1–LEC-11: Structural features and sugar-binding properties

Galectins form a large family of β-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2–5 and 8–11) from a λZAP cDNA library. Among them, lec-2–5 were found to encode 31–35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8–11 were found to encode 16–27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1–4, -6, and 8–10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various β-galactosides, i.e., N-acetyllactosamine (Galβ1-4GlcNAc), lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and asialofetuin, demonstrated that LEC-1–4, -6, and -10 have a significant affinity for β-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galβ1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.     

Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C₁₆. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to ‘lipid raft’ like membranes, suggesting that the protein could be involved in raft assembly.     

Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na⁺. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl₂, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The K_m for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.     

Model of the outer membrane potential generation by the inner membrane of mitochondria.

Voltage-dependent anion channels in the outer mitochondrial membrane are strongly regulated by electrical potential. In this work, one of the possible mechanisms of the outer membrane potential generation is proposed. We suggest that the inner membrane potential may be divided on two resistances in series, the resistance of the contact sites between the inner and outer membranes and the resistance of the voltage-dependent anion channels localized beyond the contacts in the outer membrane. The main principle of the proposed mechanism is illustrated by simplified electric and kinetic models. Computational behavior of the kinetic model shows a restriction of the steady-state metabolite flux through the mitochondrial membranes at relatively high concentration of the external ADP. The flux restriction was caused by a decrease of the voltage across the contact sites and by an increase in the outer membrane potential (up to +60 mV) leading to the closure of the voltage-dependent anion channels localized beyond the contact sites. This mechanism suggests that the outer membrane potential may arrest ATP release through the outer membrane beyond the contact sites, thus tightly coordinating mitochondrial metabolism and aerobic glycolysis in tumor and normal proliferating cells.     

Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima

The hypothesis that the primary Na+-pump, Na+-ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5-8.0. On the assumption that the ATP-hydrolysis at alkaline pH values is related to a Na+-ATPase activity, we investigated two ATP-dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Deltapsi) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5-8.0. The ATP-dependent Na+ accumulation was stimulated by the permeant NO3- anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Deltapsi generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+-ATPase of the P-type, functions in the PM of marine microalga D. maritima.     

Magnetic and structural studies of novel tetranickel hydroxamates

The dinickel(II) compound [Ni₂(μ-OAc)₂(OAc)₂(μ-H₂O)(asy·dmen)₂]·2.5H₂O, 1; undergoes facile reaction in a 1:2 molar ratio with benzohydroxamic acid (BHA) in ethanol to give the novel nickel(II) tetranuclear hydroxamate complex [Ni₄(μ-OAc)₃(μ-BA)₃(asy·dmen)₃][OTf]₂·H₂O, 2, in which the bridging acetates, bridging two nickel atoms in 1, undergo a carboxylate shift from the μ₂-η¹:η¹ bridging mode of binding to the μ₃-η¹:η² bridging three nickel atoms in the tetramer. The structure of complex 2 was determined by single-crystal X-ray crystallography. The two monodentate acetates, water and two bidentate bridging acetates of two moles of complex 1 are replaced by three monodentate bridging acetates and three benzohydroxamates. Three nickel atoms in the tetramer, Ni(2), Ni(3) and Ni(4) are in a N₂O₄ octahedral environment, while the fourth nickel atom Ni(1) is in an O(6) octahedral environment. The Ni-Ni separations are Ni(1)-Ni(2) = 3.108 Å, Ni(1)-Ni(3) = 3.104 Å and Ni(1)-Ni(4) = 3.110 Å, which are longer than previously studied in dinuclear urease inhibited models but shorter than in the nickel(II) tetrameric glutarohydroxamate complex [Ni₄(μ-OAc)₂(μ-gluA₂)₂(tmen)₄][OTf]₂, isolated and characterized previously in this laboratory. Magnetic studies of the tetrameric complex show that the four Ni(II) ions are ferromagnetically coupled, leading to a total ground spin state S_T = 4. Three analogous tetranuclear nickel hydroxamates were prepared from AHA and BHA and the appropriate dinuclear complex with either sy·dmen or asy·dmen as capping ligands.     

Caveolin-1 affects serotonin binding and cell surface levels of human 5-HT7(a) receptors

Studies using HeLa cells expressing 5-HT7 receptors showed that detergent resistant membranous lipid rafts purified by sucrose gradient centrifugation contained 5-HT7 receptors and caveolin-1. Caveolin-1 siRNA-mediated knockdown or serotonin (5-HT) treatment caused significant reductions of maximum [3H]5-HT binding to 5-HT7 receptors and total immunoreactivity of membranous 5-HT7 receptors in intact cells. Co-treatment with 5-HT, caveolin-1 siRNA and methyl-beta-cyclodextrin had no additive effects on reducing maximum binding of [3H]5-HT to 5-HT7 receptors. 5-HT-mediated reduction of [3H]5-HT binding to 5-HT7 receptors was counteracted by genistein, but not by sucrose. Caveolin-1, specifically localized in cholesterol-enriched lipid rafts, appears to regulate constitutive and agonist-stimulated cell surface levels of 5-HT7 receptors via a clathrin-independent mechanism.     

NH(4)(+)-stimulated low-K(+) uptake is associated with the induction of H(+) extrusion by the plasma membrane H(+)-ATPase in sorghum roots under K(+) deficiency

The effect of external inorganic nitrogen and K⁺ content on K⁺ uptake from low-K⁺ solutions and plasma membrane (PM) H⁺-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4 mM K⁺ and inorganic nitrogen as NO₃^-, NO₃^-/NH₄⁺ or NH₄⁺ and then starved of K⁺ for 24, 48 and 72 h. NH₄⁺ in full nutrient solution significantly affected the uptake efficiency and accumulation of K⁺, and this effect was less pronounced at the high K⁺ concentration. In contrast, the translocation rate of K⁺ to the shoot was not altered. Depletion assays showed that plants grown with NH₄⁺ more efficiently depleted the external K⁺ and reached higher initial rates of low-K⁺ uptake than plants grown with NO₃^-. One possible influence of K⁺ content of shoot, but not of roots, on K⁺ uptake was evidenced. Enhanced K⁺-uptake capacity was correlated with the induction of H⁺ extrusion by PM H⁺-ATPase. In plants grown in high K⁺ solutions, the increase in the active H⁺ gradient was associated with an increase of the PM H⁺-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2 mM K⁺, only the initial rate of H⁺-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H⁺-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO₃^--grown plants. The results suggest that K⁺ homeostasis in NH₄⁺-grown sorghum plants may be regulated by a high capacity for K⁺ uptake, which is dependent upon the H⁺-pumping activity of PM H⁺-ATPase.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.     

Biophysics of sphingolipids II. Glycosphingolipids: An assortment of multiple structural information transducers at the membrane surface

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.     

Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation

The Saccharomyces cerevisiae MID1 gene product (Mid1) is a stretch-activated Ca(2+)-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H1 to H3 (Mid1(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca(2+)-uptake activity of the mid1 mutant, whereas Mid1(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta3-22)-GFP lacking the H1 region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H1 localized in the plasma membrane in the presence of alpha-factor, suggesting that H1 is a signal sequence responsible for the alpha-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12.     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with d-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

Macromolecular crowding at membrane interfaces: adsorption and alignment of membrane peptides

Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH₄, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.