A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca²⁺-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.     

In vitro reconstitution of substrate S-acylation by the zDHHC family of protein acyltransferases

Protein S-acylation, more commonly known as protein palmitoylation, is a biological process defined by the covalent attachment of long chain fatty acids onto cysteine residues of a protein, effectively altering the local hydrophobicity and influencing its stability, localization and overall function. Observed ubiquitously in all eukaryotes, this post translational modification is mediated by the 23-member family of zDHHC protein acyltransferases in mammals. There are thousands of proteins that are S-acylated and multiple zDHHC enzymes can potentially act on a single substrate. Since its discovery, numerous methods have been developed for the identification of zDHHC substrates and the individual members of the family that catalyse their acylation. Despite these recent advances in assay development, there is a persistent gap in knowledge relating to zDHHC substrate specificity and recognition, that can only be thoroughly addressed through in vitro reconstitution. Herein, we will review the various methods currently available for reconstitution of protein S-acylation for the purposes of identifying enzyme–substrate pairs with a particular emphasis on the advantages and disadvantages of each approach.     

The reconstitution and activity of the small multidrug transporter EmrE is modulated by non-bilayer lipid composition

The ability of multidrug transport proteins within biological membranes to recognise a diverse array of substrates is a fundamental aspect of antibiotic resistance. Detailed information on the mechanisms of recognition and transport can be provided only by in vitro studies in reconstituted bilayer systems. We describe the controlled, efficient reconstitution of the small multidrug transporter EmrE in a simple model membrane and investigate the effect of non-bilayer lipids on this process. Transport activity is impaired, in line with an increase in the lateral pressure within the bilayer. We demonstrate the potential of this lateral pressure modulation method as a general approach to the folding and assembly of membrane proteins in vitro, by recovering functional transporter from a partly denatured state. Our results highlight the importance of optimising reconstitution procedures and bilayer lipid composition in studies of membrane transporters. This is particularly pertinent for multidrug proteins, and we show that the use of a sub-optimal lipid bilayer environment or reconstitution method could lead to incorrect information on protein activity.     

Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane

Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein-depleted ghosts, or detergent-treated ghosts. D-glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein-depleted ghosts, the selective solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC-720 extracts could be prepared in which the glucose transporter retained activity for days at 4 degrees C. These extracts were applied to affinity chromatography matrices of phloretin-Agarose, prepared by coupling phloretinyl-3'-benzylamine (PBA) to CH-Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4.5, from the Affigel affinity medium.     

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.     

Nanodiscs: A toolkit for membrane protein science

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self‐assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native‐like bilayer environment that maintain a target''s functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.     

Curvature-based sorting of eight lipid types in asymmetric buckled plasma membrane models

Curvature is a fundamental property of biological membranes and has essential roles in cellular function. Bending of membranes can be induced by their lipid and protein compositions, as well as peripheral proteins, such as those that make up the cytoskeleton. An important aspect of membrane function is the grouping of lipid species into microdomains, or rafts, which serve as platforms for specific biochemical processes. The fluid mosaic model of membranes has evolved to recognize the importance of curvature and leaflet asymmetry, and there are efforts toward evaluating their functional roles. This work investigates the effect of curvature on the sorting of lipids in buckled asymmetric bilayers containing eight lipid types, approximating an average mammalian plasma membrane, through coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field. The simulations reveal that 1) leaflet compositional asymmetry can induce curvature asymmetry, 2) lipids are sorted by curvature to different extents, and 3) curvature-based partitioning trends show moderate to strong correlations with lipid molecular volumes and head to tail bead ratios, respectively. The findings provide unique insights into the role of curvature in membrane organization, and the curvature-based sorting trends should be useful references for later investigations and potentially interpreting the functional roles of specific lipids.     

Evidence for chromophore-chromophore interactions in the Purple Membrane from reconstitution experiments of the chromophore-free membrane

We recently presented evidence showing that the visible CD spectrum of the purple membrane from Halobacterium halobium consists of two contributions: a broad positive band centered at the absorption maximum due to the interaction of the chromophore with the protein to which it is bound, and an exciton coupling band due to the interaction between chromophores of adjacent bacteriorhodopsin molecules in the hexagonal surface lattice (Heyn et al., 1975). This interpretation receives strong support from the present experiments in which the chromophore-free membrane is reconstituted by the addition of retinal. Since the coupling signal arises from the interaction between pairs of neighboring chromophores, its contribution to the spectrum would be expected to be very small in the initial stages of the titration experiment, but increasing quadratically with the percentage reconstitution. The broad positive band, on the other hand, is expected to increase linearly with the percentage reconstitution. On the basis of these considerations a satisfactory explanation of the CD reconstitution experiments could be given. Since it appears to be impossible to explain the titration experiments without the quadratic term, we conclude that chromophore-chromophore interactions play an important role. No significant changes in secondary structure upon reconstitution could be detected consistent with our binding model which neglects cooperativity.Abbreviations CD circular dichroism - UV ultraviolet     

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.     

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

Abstract

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.     

Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research

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S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.     

Functional reconstitution of photosystem I reaction center from cyanobacteriumSynechocystis sp PCC6803 into liposomes using a new reconstitution procedure

Photosystem I reaction center from the cyanobacteriumSynechocystis sp PCC6803 was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. Liposomes prepared by reversephase evaporation were treated with various amounts of different detergents and protein incorporation was analyzed at each step of the solubilization process. After detergent removal the activities of the resulting proteoliposomes were measured. The most efficient reconstitution was obtained by insertion of the protein complex into preformed liposomes destabilized by saturating amounts of octylglucoside. In the presence of N-methylphenazonium methosulfate and ascorbic acid, liposomes containing the reaction center catalyzed a light-dependent net H⁺ uptake as measured by the 9-aminoacridine fluorescence quenching and the pH meter. An important benefit of the new reconstitution procedure is that it produces a homogeneous population of large-size proteoliposomes with a low ionic permeability and with a majority inwardly directed H⁺ transport activity. In optimal conditions, a light-induced pH of about 1.8 units could be sustained at 20C in the presence of valinomycin. In the absence of valinomycin, a back-pressure effect of an electrical transmembrane potential decreased both the rate and the extent of the H⁺ transport. The reaction center was also co-reconstituted with F₀F₁ H⁺-ATPases from chloroplasts and from the thermophilic bacterium, PS3. The coreconstituted system was shown to catalyze a light-dependent phosphorylation which could only be measured in the presence of a high concentration of PSI (low lipid/PSI ratios) while no pH could be detected.     

Model membrane platforms to study protein-membrane interactions

Abstract

Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Purification and reconstitution of functional human P-glycoprotein

The overexpression of the P-glycoprotein, theMDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [³H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.     

Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.     

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.     

Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1

Synaptotagmin-1 (Syt1) functions as the Ca²⁺ sensor in neuronal exocytosis, and it is routinely incorporated into lipid bilayers along with other components of the fusion machinery in order to reconstruct the in vivo fusion process. Here, we demonstrate that the detergent used to reconstitute full-length Syt1 has a significant effect on the state of the protein in bilayers. When octyl-β-d -glucopyranoside is used to reconstitute the protein, Syt1 is present in an aggregated state that is mediated by the long juxta-membrane linker. EPR spectra from spin labels in the two C2 domains of Syt1 no longer resemble those obtained from a soluble construct containing these domains, and the C2B domain no longer exhibits a Ca²⁺-dependent membrane insertion. In contrast, when reconstituted using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Syt1 is largely monomeric and the EPR spectra from C2A and C2B resemble those of the soluble construct. This result demonstrates that the choice of detergent used to reconstitute Syt1 can modulate the state of the neuronal Ca²⁺-sensor.