Reconstitution of phospholipid translocase activity with purified Drs2p,a type-IV P-type ATPase from budding yeast

Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg²⁺-ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase.     

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions

Ca²⁺-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca²⁺-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P₂ in acceptor liposomes and was independent of Ca²⁺, but Ca²⁺ dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.     

Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

Sec17 [soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17^L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17^F21S,M22S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17^F21S,M22S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.Intracellular vesicular traffic between organelles is the basis of cell growth, hormone secretion, and neurotransmission. At each step of exocytic and endocytic trafficking, membranes dock and fuse, mixing their lipids and luminal contents while keeping them separate from the cytosol. Families of proteins, conserved from yeast to humans, mediate docking and fusion. Fusion requires Rab family GTPases and “effector” proteins that bind to a Rab in its active, GTP-bound state (1). Among the effectors are large, organelle-specific tethering complexes. Fusion requires SNARE proteins and their chaperones. SNAREs (2) are proteins that can “snare” (bind to) each other, in cis (when anchored to the same membrane) or in trans (when anchored to apposed, tethered membranes). SNAREs have a conserved “SNARE domain” with a characteristic heptad repeat. SNAREs are categorized as R-SNAREs if they have a central arginyl residue, or Qa-, Qb-, or Qc-SNAREs with a central glutamyl residue (3). SNAREs form RQaQbQc quaternary cis- or trans-SNARE complexes, which bind SNARE chaperones, including the Sec1/Munc18 family of SNARE binding proteins, and Sec18/NSF (N-ethylmaleimide–sensitive factor), an AAA family ATPase that drives SNARE complex disassembly (4). Sec17/α-SNAP (soluble NSF attachment protein) is a cochaperone to Sec18 that enhances its rate of SNARE complex disassembly (5).We study fusion with yeast vacuoles. The homotypic fusion of vacuoles has been studied extensively through genetic identification of vacuole morphology (vam) mutants (6) and vacuole protein sorting (vps) mutants (7), through a colorimetric assay of the fusion of isolated vacuoles (8), and more recently through the fusion of proteoliposomes reconstituted with defined, purified proteins and lipids (9–11). Sec17, Sec18, and ATP catalyze the first stage of vacuole fusion, in which cis-SNARE complexes are disassembled (12). Tethering is then supported by the Rab Ypt7 and the large, multisubunit tethering complex termed HOPS (13). Vps33, one of the HOPS subunits, is the vacuolar SM (Sec1/mUNC-18 family) protein. HOPS has direct affinity for vacuolar SNAREs (14–16), and helps to catalyze SNARE complex assembly and the subsequent fusion (17).During in vitro fusion incubations, most Sec17 is released from vacuoles during cis-SNARE complex disassembly (12). However, a few percent of the vacuolar SNAREs form trans-SNARE complexes (18), and Sec17 is a major constituent of these complexes (19). Furthermore, although Sec17 and Sec18 can disassemble trans-SNARE complexes (19) and will block fusion events in which tethering is supplied by an unphysiological agent such as polyethylene glycol (13), Sec17 and Sec18 work synergistically with HOPS to promote fusion (9, 20). This synergy is even seen when the SNAREs are initially disposed with the R-SNARE on one set of proteoliposomes and the three Q-SNAREs on the others (9), a condition that per se does not require cis-SNARE complex disassembly by Sec18. Finally, added Sec17 restores fusion to vacuoles where fusion is blocked by a defined C-terminal truncation in the SNARE domain of the Qc-SNARE Vam7, in the apparent absence of ATP or Sec18 activity (21). These observations prompted us to reevaluate the roles of Sec17 and Sec18 in the fusion pathway.We now exploit proteoliposomes bearing purified vacuolar Rab and SNAREs to reinvestigate the roles of Sec17 and Sec18. Generous amounts of Sec18 alone can disassemble cis-SNARE complexes, allowing proteoliposomes bearing all four vacuolar SNAREs to fuse at a moderate rate. The rate of fusion can be stimulated by wild-type Sec17, as expected, but also by a Sec17 mutant that has greatly diminished capacity to stimulate Sec18. This suggested that Sec17 could act in ways other than through Sec18 stimulation. We therefore examined fusion incubations where the SNAREs are disposed on complementary proteoliposomes such that Sec18 is not required at all. We find that Sec17 can trigger rapid fusion of proteoliposomes that are already joined by trans-SNARE associations.     

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms.Membrane fusion mediates a variety of biological processes, such as fertilization and cell growth, hormone secretion, neurotransmission, nutrient uptake, and viral infection (1). Vesicle trafficking between organelles, a major tool for intracellular transport of materials, is also regulated by membrane fusion: Membrane fusion between transport vesicles and target compartments releases the cargo stored in the vesicles into the lumen of the compartments. To maintain the unique chemical environment of each organelle, biological membrane fusion occurs with spatiotemporal precision but without leakage of the luminal contents. This nature of biological membrane fusion may be achieved through the cooperation of various proteins, such as Rab GTPases and their effectors, SNARE [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] proteins, and SNARE chaperones (2, 3). SNARE proteins bring two membrane bilayers into close proximity, which promotes the fusion of the apposed membranes (4, 5). The molecular mechanism by which SNARE proteins mediate membrane fusion has been intensively studied in synaptic vesicle fusion, which mediates neurotransmission at the synapse, whereby neurotransmitters released by presynaptic neurons are recognized by their receptors on postsynaptic neurons (6, 7). Depolarization of presynaptic nerve terminals by an action potential opens Ca²⁺ channels in the presynaptic membrane. Ca²⁺ influx into the presynaptic cell then triggers membrane fusion between the presynaptic plasma membrane and synaptic vesicles, leading to the release of neurotransmitter. Synaptic vesicle fusion is mediated by three neuronal SNARE proteins: syntaxin, SNAP25, and synaptobrevin (also referred to as VAMP). Synaptobrevin and syntaxin-1 each contain one SNARE motif, whereas SNAP25 contains two. One SNARE motif from synaptobrevin (v-SNARE) on a synaptic vesicle and three SNARE motifs provided by syntaxin-1 and SNAP25 (t-SNAREs) from the plasma membrane assemble into a tight trans-SNARE complex that brings the two membranes into close apposition. This close apposition, in turn, induces lipid bilayer merging, thus releasing neurotransmitter into the synaptic cleft.In addition to the neuronal SNARE proteins, many other regulatory proteins, such as Munc18 and synaptotagmin, are required for synaptic vesicle fusion in vivo (8, 9). Munc18, a member of the Sec1/Munc18 (SM) protein family, seems to play a variety of roles in synaptic vesicle fusion. First, Munc18 binds to free syntaxin molecules and keeps them in a closed, inactive state, helping to prevent the formation of premature SNARE complexes (10, 11). Additionally, Munc18 interacts with the syntaxin molecule within assembled t-SNARE complexes, guiding them in a manner conducive to productive trans-SNARE complex formation, which triggers membrane fusion (12). Neuronal synaptotagmin, anchored to synaptic vesicles, functions as a calcium sensor for synaptic vesicle fusion (9, 13, 14). Although its mode of action is not yet fully defined, calcium binding to synaptotagmin triggers synaptic vesicle fusion. Despite the importance of these and many other proteins for synaptic vesicle fusion, the three neuronal SNARE proteins are thought to constitute the minimal components sufficient to drive membrane fusion: On their own, they can induce fusion between proteoliposomes carrying both syntaxin and SNAP25 and those reconstituted with synaptobrevin (15). Although these reconstitution experiments strongly support the concept that neuronal SNARE proteins suffice to induce membrane fusion, it is still unclear whether they can also induce fusion between biological membranes for the following reasons: (i) Liposome fusion, unlike biological membrane fusion, is intrinsically promiscuous: Even protein-free liposomes can fuse under certain conditions (16). (ii) Liposome fusion assays often rely on detection of lipid mixing, which can occur without content mixing. Liposome rupture (17) or clustering without fusion (18) can generate false-positive signals. (iii) Finally, because detergent is used to reconstitute SNARE proteins into liposomes, the potential presence of residual detergent, which affects the integrity of liposomes and their lipid mixing, cannot be completely excluded.Homotypic yeast vacuole fusion has been used to study membrane fusion mechanisms (19–21). In vitro assays using isolated yeast vacuoles have been established that measure the mixing of vacuole luminal compartments (22, 23). The fusion of isolated yeast vacuoles in vitro is mediated by evolutionarily conserved membrane fusion machinery, which involves not only SNARE proteins but also Rab and SM proteins. In this study, to address whether neuronal SNARE proteins are sufficient to induce biological membrane fusion, we engineered the budding yeast Saccharomyces cerevisiae to express neuronal SNARE proteins in vacuoles. Using these yeast vacuoles, we then show that neuronal SNARE proteins can induce vacuole fusion in a Rab- and SM protein-independent manner.     

Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H⁺)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H⁺)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.     

Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome

The mammalian innate immune system uses Toll-like receptors (TLRs) and Nod-LRRs (NLRs) to detect microbial components during infection. Often these molecules work in concert; for example, the TLRs can stimulate the production of the proforms of the cytokines IL-1β and IL-18, whereas certain NLRs trigger their subsequent proteolytic processing via caspase 1. Gram-negative bacteria use type III secretion systems (T3SS) to deliver virulence factors to the cytosol of host cells, where they modulate cell physiology to favor the pathogen. We show here that NLRC4/Ipaf detects the basal body rod component of the T3SS apparatus (rod protein) from S. typhimurium (PrgJ), Burkholderia pseudomallei (BsaK), Escherichia coli (EprJ and EscI), Shigella flexneri (MxiI), and Pseudomonas aeruginosa (PscI). These rod proteins share a sequence motif that is essential for detection by NLRC4; a similar motif is found in flagellin that is also detected by NLRC4. S. typhimurium has two T3SS: Salmonella pathogenicity island-1 (SPI1), which encodes the rod protein PrgJ, and SPI2, which encodes the rod protein SsaI. Although PrgJ is detected by NLRC4, SsaI is not, and this evasion is required for virulence in mice. The detection of a conserved component of the T3SS apparatus enables innate immune responses to virulent bacteria through a single pathway, a strategy that is divergent from that used by plants in which multiple NB-LRR proteins are used to detect T3SS effectors or their effects on cells. Furthermore, the specific detection of the virulence machinery permits the discrimination between pathogenic and nonpathogenic bacteria.     

Increased efficiency of charge-mediated fusion in polymer/lipid hybrid membranes

Due to their augmented properties, biomimetic polymer/lipid hybrid compartments are a promising substitute for natural liposomes in multiple applications, but the protein-free fusion of those semisynthetic membranes is unexplored to date. Here, we study the charge-mediated fusion of hybrid vesicles composed of poly(dimethylsiloxane)-graft-poly(ethylene oxide) and different lipids and analyze the process by size distribution and the mixing of membrane species at μm and nano scales. Remarkably, the membrane mixing of oppositely charged hybrids surpasses by far the degree in liposomes, which we correlate with properties like membrane disorder, rigidity, and ability of amphiphiles for flip-flop. Furthermore, we employ the integration of two respiratory proteins as a functional content mixing assay for different membrane compositions. This reveals that fusion is also attainable with neutral and cationic hybrids and that the charge is not the sole determinant of the final adenosine triphosphate synthesis rate, substantiating the importance of reconstitution environment. Finally, we employ this fusion strategy for the delivery of membrane proteins to giant unilamellar vesicles as a way to automate the assembly of synthetic cells.

In eukaryotic cells, membrane fusion by SNARE proteins (1) plays a crucial role in various cellular functions, such as exo- and endocytosis, membrane remodeling, cell division, signal transduction, and intracellular trafficking. In addition to fusogenic proteins, Ca²⁺ is another important mediator that usually participates in fusion events together with SNAREs; e.g., Ca²⁺ triggers neurotransmitter release during the exocytosis of synaptic vesicles (2). Accompanying players in many charge-related mechanisms are also anionic phospholipids like phosphatidylinositol and phosphatidylserine (PS) (3); e.g., the redistribution of PS in the outer leaflet takes place during apoptosis and syncytial fusion (4). In addition to fusion occurring inside the same organism, viruses have developed their own ways to insert genetic material into the host cell (5). All these natural fusion facilitators offer a diverse toolbox that can be utilized in bottom-up synthetic biology to mimic some of the aforementioned functions. The mechanisms behind salt (6–9), charge (10–12), SNARE (13, 14), and viral (15, 16) fusion of phospholipid compartments have been well characterized and employed for the construction of lifelike functional systems. In this way, integral membrane proteins have been co-reconstituted by SNAREs (17) or delivered to liposomes via electrostatic attraction (18, 19), while Mg²⁺ has facilitated the automated formation of droplet-stabilized protocells in microfluidics (20).Liposomes are essentially reductionist models of natural cells and their membranes. Hence, they serve as workhorses to study membrane proteins (MPs) and interfacial phenomena in general but also as a chassis for the construction of artificial organelles and cells. However, without the natural metabolism for membrane replenishment, the limited chemical stability of liposomes poses limitations. This liability arises from the fact that their building blocks are prone to oxidation and hydrolysis, which may cause destabilization of the membrane and, consequently, leakage of cytosolic cargo and deactivation of the inserted MPs (21–23). To circumvent this issue, lipids have been completely or partially substituted with synthetic copolymers (24–27) to form polymersomes or hybrids, respectively. The latter mixing strategy uses the synergistic effect of both types of amphiphiles (28) and is especially useful when the polymer environment alone does not well suit certain MPs (24). Polymers may thereby not only contribute to enhanced functional and structural durability but also can endow direct protection against oxidative damage when the synthetic amphiphile dominates the composition (21). Furthermore, the reorganization of hybrid membranes upon protein insertion has been shown to reduce the proton permeability in comparison to proteoliposomes (21) and has been used for controlled formation of raft-like domains (29). Not only polymers but also dendrimers have been used for the generation of hybrids due to matching thickness, chemical programmability, and excellent stability of the resulting membranes. Thus, their coassembly with bacterial (30) or human cell membranes (31) has offered platforms for MP reconstitution, glycosylation, and cell adhesion. Finally, note that the concept of amphiphile augmentation is not limited to vesicles and has been used in other model systems for MP reconstitution such as nanodiscs (32). When discussing the advantages that polymers bring, the latter are often juxtaposed with lipids with respect to the different thickness and permeability, leading to better mechanical stability. However, the versatility of synthetic amphiphiles allows for the modulation of the mechanical properties by the respective architectures, chemistries, and molar ratios—i.e., polymer membranes are not rigid by default but tunable. This is evident, for instance, in the case of phase separation (33, 34), which is governed by the hydrophobic mismatch between lipids and polymers, and the resulting line tension (35). Thus, changing only the copolymer architecture from triblock to grafted while keeping the building blocks and membrane thickness the same may suffice for more efficient mixing and formation of fewer domains (36).As discussed above, fusion is one of the most widespread mechanisms for cellular trafficking and therefore represents the most intuitive tool for the integration of functional parts in artificial cells, regardless of the membrane chemistry. In this respect, we recently succeeded in employing SNARE machinery for the fusion of hybrid vesicles made of phosphatidylcholine and poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) (37). Meanwhile, we were unable to fuse these hybrids in the absence of fusogenic proteins; the agitation in salt solutions that induced the successful fusion of pure polymersomes caused only fission when lipids were present (38). To the best of our knowledge, no other attempts for protein-free fusion of mixed polymer/lipid vesicles have been made to date. Therefore, herein we study the fusion of PDMS-g-PEO–based hybrids mediated by oppositely charged lipids. We characterize the process by following the evolution of the size distribution, next to membrane and content mixing, whereby the latter is assessed by the functional integration of two respiratory enzymes. Furthermore, we employ this electrostatic cue to overcome the fusion barrier in compartments with low curvature. To this end, we monitor the membrane mixing between micro- and nanocompartments via fluorescence microscopy, whereas cryoelectron microscopy (cryoEM), surface charge, flip-flop, and membrane disorder measurements provide further insights into the role of the synthetic amphiphile. Finally, we apply the charge-mediated fusion of hybrids as a controlled delivery mechanism in a microfluidic setup.     

Solubilization of a calcium dependent adenosine triphosphatase from rat heart sarcolemma

The rat heart sarcolemmal ATPase activity was found to be dependent on the presence of Ca²⁺ (v_max = 37 μmol Pi/mg protein/h and K_a = 0.59 mm) or Mg²⁺ ions (v_max = 28 μmol Pi/mg protein/h and K_a = 0.87 mm). The incubation of sarcolemmal membrane with trypsin stimulated the Ca²⁺ dependent ATPase activity without affecting the Mg²⁺ dependent ATPase activity. The increase in Ca²⁺ dependent ATPase activity was associated with a decrease in the K_a value from 0.59 to 0.45 mm and an increase in v_max from 37 to 69 μmol Pi/mg protein/h. In membrane preparations treated with detergents, trypsin decreased the Mg²⁺ dependent ATPase activity whereas the Ca²⁺ dependent ATPase activity was increased. Trypsin treatment of sarcolemma released proteins in the supernatant that showed ATP hydrolysis in the presence of Ca²⁺ but not Mg²⁺; while the residual membrane showed an ATPase dependent on Ca²⁺ (v_max = 67 μmol Pi/mg protein/h and K_a = 0.58 mm) and Mg²⁺ (v_max = 58 μmol Pi/mg protein/h and K_a = 0.87 mm). The proteins released in the supernatant were subjected to column chromatography on sepharose-6B, and DEAE cellulose and a Ca²⁺-dependent ATPase activity (v_max = 200 μmol Pi/mg protein/h and K_a = 0.25 mm) was recovered as a distinct peak. These results indicate solubilization of a Ca²⁺ dependent ATPase which, unlike the enzyme present in heart sarcolemma, showed negligible activation by Mg²⁺.     

Electrophysiological characterization of LacY

Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H⁺ symport into the proteoliposomes generates transient currents. Studies at different lipid-to-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H⁺ symport is H⁺ release. In contrast, LacY mutants Glu-325→Ala and Cys-154→Gly, which bind ligand normally, but are severely defective with respect to lactose/H⁺ symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H⁺ symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H⁺ during downhill sugar/H⁺ symport, which is the limiting step for this mode of transport.     

Fusion of liposomes with mitochondrial inner membranes

A procedure is outlined for the fusion of mixed phospholipid liposomes (small unilamellar vesicles) with the mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was initiated by manipulation of the pH of a mixture of freshly sonicated liposomes and the functional inner membrane/matrix fraction of rat liver mitochondria. During the pH fusion procedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron microscopy. After the pH fusion procedure, a number of ultrastructural, compositional, and functional characteristics were found to be proportionally related: the membrane surface area increased; the lateral density distribution of intramembrane particles (integral proteins) in the plane of the membrane decreased whereas the particles remained random; the membrane became more buoyant; the ratio of membrane lipid phosphorus to total membrane protein increased; the ratio of membrane lipid phosphorus to heme a of cytochrome c oxidase increased; and the rate of electron transfer between some interacting membrane oxidoreduction proteins decreased. These data reveal that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) and that integral membrane proteins diffused freely into the laterally expanding bilayer. Furthermore, the data suggest that the rate of electron transfer may be limited by the rate of lateral diffusion of oxidoreduction components in the bilayer of the mitochondrial inner membrane.     

Mg/KCl-ATPase of plant plasma membranes is an electrogenic pump

The function of the Mg²⁺-requiring KCl-stimulated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of higher plants in active ion transport was investigated by using a purified microsomal fraction containing sealed plasma membrane vesicles. (Sze, H. (1980) Proc. Natl. Acad. Sci. USA 77, 5904-5908). A transmembrane electrical potential (+30 to +44 mV), monitored by uptake of a permeant anion (³⁵SCN^-), was generated specifically by ATP in purified microsomal vesicles of tobacco callus. ATP-dependent ³⁵SCN^- uptake required Mg²⁺, was optimal at pH 6.75, and showed similar ATP concentration dependence as the Mg²⁺-requiring KCl-stimulated ATPase activity. Plasma membrane ATPase inhibitors (N,N′-dicyclohexylcarbodiimide and vanadate) inhibited generation of the ATP-dependent electrical potential. A proton conductor (carbonyl cyanide m-chlorophenylhydrazone), but not a K⁺ ionophore (valinomycin), completely collapsed the electrical potential. The results provide in vitro evidence that the Mg²⁺/KCl-ATPase of higher plants is an electrogenic pump. These results are consistent with the hypothesis that an electrogenic H⁺ pump is catalyzed by the plasma membrane ATPase of plants.     

Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development

Magnesium (Mg²⁺) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg²⁺ transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg²⁺ influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg²⁺. Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg²⁺ concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg²⁺ or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg²⁺ transport system.     

Application of Liposome Encapsulating Lactobacillus curvatus Extract in Cosmetic Emulsion Lotion

Probiotic extracts have various positive attributes, such as antioxidant, tyrosinase inhibitory, and antimicrobial activity. Lactobacillus curvatus produces bacteriocin, which activates the lipid membrane structure and has potential as a natural preservative for cosmetic emulsions. In this study, L. curvatus extract was encapsulated in liposomes and formulated as an oil-in-water (O/W) emulsion. Radical scavenging activity, tyrosinase inhibition, and challenge tests were conducted to confirm the liposome activity and the activity of the applied lotion emulsion. The liposome-encapsulated extract had a relatively high absolute ζ-potential (52.53 > 35.43), indicating its stability, and 96% permeability, which indicates its potential as an active agent in lotion emulsions. Characterization of emulsions containing the liposomes also indicated a stable state. The liposome-encapsulated extract exhibited a higher radical scavenging activity than samples without the extract and non-encapsulated samples, and the functionality was preserved in the lotion emulsion. The tyrosinase inhibition activity of the lotion emulsion with the liposome-encapsulated extract was similar to that of the non-treated extract. Candida albicans and Aspergillus niger were also inhibited in the challenge test with the lotion emulsions during storage. Collectively, these findings indicate that the liposome-encapsulated extract and the lotion containing the encapsulated extract have potential applicability as natural preservatives.     

Partial characterization of a phosphorylated intermediate associated with the plasma membrane ATPase of corn roots

The phosphorylated protein associated with a deoxycholate-extracted plasma membrane fraction from corn (Zea mays L. var WF9 × Mol7) roots was characterized in order to correlate its properties with those of plasma membrane ATPase. Its phosphorylation, like that of plasma membrane ATPase, was dependent on Mg²⁺, substrate specific for ATP, insensitive to azide, oligomycin, or molybdate, and sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, or vanadate. Monovalent cations affected the phosphorylation of the protein in a manner consistent with their stimulatory effects on ATPase. For K⁺, this was shown to occur through an increase in the turnover of the phosphoenzyme. Analysis of the phosphorylated protein by NaDodSO₄/polyacrylamide gel electrophoresis revealed the presence of a single labeled polypeptide with a molecular weight of about 100,000. Phosphorylation of this polypeptide was dependent on Mg²⁺, sensitive to K⁺, and inhibited by vanadate. It is concluded that this polypeptide represents the catalytic subunit of the plasma membrane ATPase. These results are discussed in terms of a model for the coupling of metabolic energy to H⁺ and K⁺ transport in higher plants.     

A repulsion mechanism explains magnesium permeation and selectivity in CorA

Magnesium (Mg²⁺) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg²⁺ uptake and is self-regulated by intracellular Mg²⁺. Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg²⁺-selective channel, fully excluding monovalent cations, and Ca²⁺, whereas in absence of Mg²⁺, CorA is essentially nonselective, displaying only mild preference against other divalents (Ca²⁺ > Mn²⁺ > Co²⁺ > Mg²⁺ > Ni²⁺). Selectivity against monovalent cations takes place via Mg²⁺ binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca²⁺ channel selectivity despite differences in sequence and overall structure.Among biological divalent cations, Mg²⁺ is not only the most abundant, but also plays an essential role in a wealth of cellular processes, including enzymatic reactions, and the stability of nucleic acids and biological membranes (1). Although the biological importance of Mg²⁺ is well established, the molecular entities and mechanisms that govern its cellular homeostasis are not well understood. In bacteria, Mg²⁺ influx is primarily catalyzed by members of the CorA family of divalent ion transport systems (2, 3). The X-ray structure of CorA has provided an excellent template toward a molecular understanding of the mechanisms underlying Mg²⁺ influx (4–7). However, although CorA has been crystallized in a wide range of conditions, so far all available CorA structures seem to correspond to nonconductive conformations, which obviously limits the basic mechanistic insights regarding Mg²⁺ selectivity and translocation that can be derived from these high-resolution structures. Computational analyses, together with NMR, X-ray absorption, and Raman spectroscopy studies, have established that Mg²⁺ holds to its first hydration shell much more tightly than any other physiological cation (8–11); this implies that any Mg²⁺-selective transport system must either compensate for the high hydration energy (and accommodate the invariable octahedral geometry of this hexacoordinated ion) or establish a selectivity mechanism able to discriminate a hydrated or partially hydrated Mg²⁺ ion from monovalent and other divalent cations.Several hypotheses have been postulated to explain CorA’s function, including its role as a Mg²⁺ -selective channel (12), a Co²⁺ transporter (13), and even as an exporter of divalent cations (14). However, detailed mechanistic evaluation of CorA’s functional properties has been limited by the resolution of existing functional assays (15). Mg²⁺ transport through CorA depends on the combination of three parameters: (i) number of open gates, (ii) the electrical potential across the membrane, and (iii) the Mg²⁺ driving force, none of which can be properly controlled with sufficient time-resolution in in vivo experiments. Although a prokaryotic membrane protein, we have been able to heterologously express CorA in Xenopus oocytes, which, in combination with standard electrophysiological approaches, allowed us to measure CorA-catalyzed divalent macroscopic currents under a variety of ionic conditions. Crystallographic studies have suggested that intracellular Mg²⁺ act as the main regulator of CorA gating under physiological conditions (6). That Mg²⁺ acts as both a gating ligand and charge carrier ultimately complicates functional studies of CorA permeation and selectivity properties. To circumvent this issue we used a mutation at the divalent cation sensor that abolishes CorA Mg²⁺-dependent gating (Fig. 1A). This construct is ideally suited to evaluate ion permeation because it stabilizes steady-state currents by inhibiting the divalent ion-driven negative-feedback loop that defines CorA gating. Our results demonstrate that CorA is a bona fide multioccupancy ion channel, and that its divalent cation permeation and tight selectivity against monovalent cations can be explained on the basis of a block and repulsion mechanism, where the canonical “signature sequence” Gly-Met-Asn (GMN) plays a central role.Open in a separate windowFig. 1.CorA-driven Mg²⁺ currents recorded from TEVC. (A) The divalent cation sensor is highlighted on a cartoon representation of CorA crystal structure. Residues Asp89 and Asp253 are shown as purple sticks (B). The membrane potential (Vm) is clamped and held at −60 mV. The external solution is exchanged between two isosmotic buffers: one containing no monovalent or divalent cation (colored in gray on the horizontal bar), and one containing 20 mM Mg²⁺ (noted Mg²⁺). A representative trace recorded on a CorA–WT-expressing oocyte is shown in teal, and control oocyte trace is shown in gray and D253K in purple. The horizontal dotted line indicates the 0 A current level. (C) Representative traces of CorA D253K mutant in TEVC. The voltage pulse protocol is shown on top of the current traces. The dotted line represents the 0 current levels. (D) The corresponding I/V relationships recorded at different external Mg²⁺ concentrations are shown. The GHK-flux equation fits are displayed as solid lines, and experimental values are dots. (E) Mg²⁺ current recorded at −60 mV under external solution perfusion. The external solution is changed stepwise and the corresponding solution exchange protocol is superimposed to the trace. (F) Mean values (±SD) of several traces (n ≥ 5) were recorded and normalized to the maximum current. The values were plotted against the external [Mg²⁺] and fitted with a single-site binding curve.     

The carboxy-terminal domain of complexin I stimulates liposome fusion

Regulated exocytosis requires tight coupling of the membrane fusion machinery to a triggering signal and a fast response time. Complexins are part of this regulation and, together with synaptotagmins, control calcium-dependent exocytosis. Stimulatory and inhibitory functions have been reported for complexins. To test if complexins directly affect membrane fusion, we analyzed the 4 known mammalian complexin isoforms in a reconstituted fusion assay. In contrast to complexin III (CpxIII) and CpxIV, CpxI and CpxII stimulated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-pin assembly and membrane fusion. This stimulatory effect required a preincubation at low temperature and was specific for neuronal t-SNAREs. Stimulation of membrane fusion was lost when the carboxy-terminal domain of CpxI was deleted or serine 115, a putative phosphorylation site, was mutated. Transfer of the carboxy-terminal domain of CpxI to CpxIII resulted in a stimulatory CpxIII-I chimera. Thus, the carboxy-terminal domains of CpxI and CpxII promote the fusion of high-curvature liposomes.     

Type VI secretion system sheaths as nanoparticles for antigen display

The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications.The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between effector cells and target cells (1–4). It is conserved in 25% of Gram-negative bacteria, including Vibrio cholerae, Pseudomonas aeruginosa and Escherichia coli. The T6SS plays a crucial role in bacterial pathogenicity and symbiosis, targeting either eukaryotic cells or competitor bacterial cells (5). The assembled and functional T6SS apparatus has structural homology to bacteriophage T4 phage tail components and can be divided into two distinct assemblies: a contractile phage tail-like structure and a transmembrane complex (6). V. cholerae VipA and VipB (named for ClpV-interacting protein A and B) and orthologous proteins in other bacteria build within the cytosol of effector cells a tubular sheath structure that is anchored to the various layers of the cell envelope through its association with the T6SS transmembrane complex (7).VipA/B sheaths are composed of six protofilaments arranged as a right-handed six-start helix similar to early T4 tail sheaths (8). Each protofilament is formed by a VipA/B heterodimer, and the atomic-resolution structure of a native contracted V. cholerae sheath has been recently determined by cryo-electron microscopy (9). Stable expression of VipB in V. cholerae requires the presence of VipA, and VipA/B heterodimers can be recruited into assembled tubular sheath structures spontaneously (10, 11). Because both ends of VipA are exposed on the external surface of the sheath tubules, a C-terminal fusion of VipA protein with superfolded green fluorescent protein (sfGFP) is functional in T6SS sheath assembly and activity, as previously demonstrated (3).Because these tubular structures are assembled in cytoplasm and can be purified from bacteria (3), we explored the possibility that T6SS sheaths could be used as a new particle-based delivery system for vaccine antigens. It is thought that particulate structures used for vaccine formulations are efficiently targeted for uptake by antigen-presenting cells (APCs) and interact directly with antigen-specific B cells generating humoral responses (12). Although particulate protein antigens may be more resistant to degradation, they are eventually proteolytically processed, and the resulting peptides are presented by the major histocompatibility complex (MHC) class I and class II molecules in a process that leads to activation of CD4⁺ and CD8⁺ T-cell helper and effector responses. Examples of particulate vaccine delivery systems include lipid-based systems [emulsions, immune-stimulating complexes (ISCOMs), liposomes, virosomes], polymer-based structures (e.g., nano-/microparticles), and virus-like particles (VLPs), with each of these systems presenting their own spectrum of advantages and disadvantages for practical use as human immunogens (13).In this work, VipA/B sheaths displaying heterologous protein antigens on the surface were generated and tested as a particulate vaccine antigen delivery system. Our results show that sheath-like structures displaying different antigens were immunogenic and that antibodies elicited against one of these, the Neisseria meningitidis factor H binding protein (fHbp), were functional in a serum bactericidal assay. The T6SS antigen delivery system demonstrates potential as a multivalent particle to deliver one or more antigens simultaneously into the same antigen-presenting cell. Moreover, the use of heterologous VipA and VipB sheaths displaying a common antigen in sequential vaccine booster regimens minimizes immune responses against the delivery system itself and focuses the immune responses against the common antigen of interest.     

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonas tolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonas agarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.