Membrane hemifusion: crossing a chasm in two leaps

During membrane fusion, the outer leaflets of the two membranes merge first, whereas the distal membrane leaflets remain separate until the opening of a fusion pore. This intermediate stage, called hemifusion, is a critical event shared by exocytosis, protein trafficking, and viral entry.     

Membrane fusion induced by neuronal SNAREs transits through hemifusion

Synaptic transmission requires the controlled release of neurotransmitter from synaptic vesicles by membrane fusion with the presynaptic plasma membrane. SNAREs are the core constituents of the protein machinery responsible for synaptic membrane fusion. The mechanism by which SNAREs drive membrane fusion is thought to involve a hemifusion intermediate, a condition in which the outer leaflets of two bilayers are combined and the inner leaflets remain intact; however, hemifusion has been observed only as an end point rather than as an intermediate. Here, we examined the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs using fluorescence assays that monitor both total lipid mixing and inner leaflet mixing. Our results demonstrate that hemifusion is dominant at the early stage of the fusion reaction. Over time, hemifusion transitioned to complete fusion, showing that hemifusion is a true intermediate. We also show that hemifusion intermediates can be trapped, likely as unproductive outcomes, by modulating the surface concentration of the SNARE proteins.     

Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin.

Fusion between influenza virus and target membranes is mediated by the viral glycoprotein hemagglutinin (HA). Replacement of the transmembrane domain of HA with a glycosylphosphatidylinositol (GPI) membrane anchor allows lipid mixing but not the establishment of cytoplasmic continuity. This observation led to the proposal that the fusion mechanism passes through an intermediate stage corresponding to hemifusion between outer monolayers. We have used confocal fluorescence microscopy to study the movement of probes for specific bilayer leaflets of erythrocytes fusing with HA-expressing cells. N-Rh-PE and NBD-PC were used for specific labeling of the outer and inner membrane leaflet, respectively. In the case of GPI-HA-induced fusion, different behaviors of lipid transfer were observed, which include 1) exclusive movement of N-Rh-PE (hemifusion), 2) preferential movement of N-Rh-PE relative to NBD-PC, and 3) equal movement of both lipid analogs. The relative population of these intermediate states was dependent on the time after application of a low pH trigger for fusion. At early time points, hemifusion was more common and full redistribution of both bilayers was rare, whereas later full redistribution of both probes was frequently observed. In contrast to wild-type HA, the latter was not accompanied by mixing of the cytoplasmic marker Lucifer Yellow. We conclude that 1) the GPI-HA-mediated hemifusion intermediate is meta-stable and 2) expansion of an aqueous fusion pore requires the transmembrane and/or cytoplasmic domain of HA.     

Atomic force microscope spectroscopy reveals a hemifusion intermediate during soluble N-ethylmaleimide-sensitive factor-attachment protein receptors-mediated membrane fusion

This study investigated the effect of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptors (SNAREs) on the fusion of egg L-α-phosphatidylcholine bilayers using atomic force microscope (AFM) spectroscopy. AFM measurements of the fusion force under compression were acquired to reveal the energy landscape of the fusion process. A single main energy barrier governing the fusion process was identified in the absence and presence of SNAREs in the bilayers. Under compression, a significant downward shift in the fusion dynamic force spectrum was observed when cognate v- and t-SNAREs were present in the opposite bilayers. The presence of vesicle-associated membrane protein (VAMP) and binary syntaxin and SNAP 25 in the apposed bilayers resulted in a reduction in the height of the activation potential by ∼1.3 k_BT and a >2-fold increase in the width of the energy barrier. The widening of the energy barrier in the presence SNAREs is interpreted as an increase in the compressibility of the membranes, which translates to a greater ease in the bilayer deformation and subsequently the fusion of the membranes under compression. Facilitation of membrane fusion was observed only when SNAREs were present in both bilayers. Moreover, addition of the soluble cytoplasmic domain of VAMP, which interferes with the interaction between opposing v- and t-SNAREs, prevented such facilitation. These observations implicated the interaction between the cytoplasmic domains of opposing SNAREs in the observed fusion facilitation, possibly by destabilizing the bilayers through pulling on their transmembrane segments. Our AFM compression measurements revealed that SNARE-mediated membrane fusion proceeded through a sequence of two ∼5 nm collapses of the membrane, an observation that is consistent with the existence of a hemifused state during the fusion process.     

Activation of initiator caspases through a stable dimeric intermediate

Structural and biochemical studies have revealed that procaspases form dimers prior to proteolytic activation. How the two procaspases interact in the dimer is unclear. To study the mechanisms of dimer-dependent caspase activation we used a heterodimeric system so that two caspase molecules can be specifically brought together. Surprisingly, only one caspase partner in the dimer needs to be enzymatically active for caspase processing and activation to occur. Caspase activation is inefficient in the dimer in the absence of intramolecular processing, suggesting that caspase activation is initiated via intramolecular processing. Homodimerization of caspase-8 or caspase-9 leads to the formation of a stable dimeric complex. However, heterodimerization between caspase-8 and caspases-3, -9, or -10 failed to induce stable dimer formation or caspase activation. Our data suggest that the formation of a stable dimeric intermediate initiates caspase activation.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

NMR assignments of a stable processing intermediate of human frataxin

Frataxin, a nuclear encoded protein targeted to the mitochondrial matrix, has recently been implicated as an iron chaperone that delivers Fe(II) to the iron-sulfur assembly enzyme ISU. During transport across the mitochondrial membrane, the N-terminal mitochondrial targeting sequence of frataxin is cleaved in a two-step process to produce the “mature” protein found within the matrix; however, N-terminally extended forms of the protein have also been observed in vivo as a result of processing deficiencies. Structural characterization studies of the mature human frataxin ortholog suggest the protein’s N-terminus is predominately unfolded, in contrast to what has been observed for the yeast ortholog. Here we report the NMR assignments of a stable intermediate in the processing of human frataxin. These studies were completed to provide structural insight into editing events that lead to mature protein formation. This report also provides structural details of frataxin editing anomalies produced in vivo during altered protein processing events.     

The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

cAMP-GEFII is a direct target of cAMP in regulated exocytosis

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.     

Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.     

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.     

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying β-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of β-glucosidase at 90°C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90°C with an overall ΔG° of ∼ 20 kcal mol⁻¹. The high temperatures needed to chemically denature P. furiosus β-glucosidase and the large ΔG° of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of β-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.     

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.     

Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis

Insulin triggers glucose uptake into skeletal muscle and adipose tissues by gaining the available number of glucose transporter 4 (GLUT4) on the cell surface. GLUT4-loaded vesicles are targeted to plasma membrane from the intracellular reservoir through multiple trafficking and fusion processes that are mainly regulated by Akt. However, it is still largely unknown how GLUT4 expression in the cell surface is promoted by insulin. In the present study, we identified tomosyn at Ser-783 as a possible Akt-substrate motif and examined whether the phosphorylation at Ser-783 is involved in the regulation of GLUT4 expression. Both Akt1 and Akt2 phosphorylated the wild-type tomosyn, but not the mutant tomosyn in which Ser-783 was replaced with Ala. Phosphorylation of tomosyn at Ser-783 was also observed in the intact cells by insulin stimulation, which was blocked by PI3K inhibitor, LY294002. In vitro pull-down assay showed that phosphorylation of tomosyn at Ser-783 by Akt inhibited the interaction with syntaxin 4. Insulin stimulation increased GLUT4 in the cell surface of CHO-K1 cells to promote glucose uptake, however exogenous expression of the mutant tomosyn attenuated the increase by insulin. These results suggest that Ser-783 of tomosyn is a target of Akt and is implicated in the interaction with syntaxin 4.     

Membrane events in adrenal chromaffin cells during exocytosis: a freeze-etching analysis after rapid cryofixation

Catecholamine-storing chromaffin cells, isolated from bovine adrenal medullae by collagenase digestion, were stimulated with carbachol at 37 degrees C; aliquots for controls were kept at 37 degrees C: Starting from this temperature, cells were ultrarapidly frozen with the use of a sandwich-propane-jet procedure and freeze-fractured. The replicas were analysed quantitatively for exocytotic activity: After stimulation the cell membrane displayed a significant increase of exo-endocytotic openings varying in size from 20 to 300 nm. The number of openings increased in parallel to the catecholamine output. At no stage could a clearing of membrane-intercalated particles (MIPs) be observed. Openings of all size classes were etchable. Results from the PF-face were comparable with those of the EF-face. We conclude that (i) exocytosis in isolated chromaffin cells starts as a focal event; the smallest possible stages are about 10 nm in size, (ii) fusion proceeds without previous rearrangement of MIPs, and (iii) the opening starts without formation of a diaphragm.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

Membrane repair: Ca(2+)-elicited lysosomal exocytosis.

Cells in exposed positions are subject to injury and therefore need membrane repair mechanisms. Ca(2+) entry inevitably follows membrane rupture and recent studies indicate that this elicits repair via Ca(2+)-activated exocytosis of lysosomes, regulated by lysosomal synaptotagmin VII.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.