During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome1-4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of auto- and deubiquitination5-8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membrane-spanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1's RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1.

The inner nuclear membrane (INM) is continuous with the endoplasmic reticulum (ER) but harbors a distinctive proteome essential for nuclear functions. In yeast, the Asi1/Asi2/Asi3 ubiquitin ligase complex safeguards the INM proteome through the clearance of mislocalized ER membrane proteins. How the Asi complex selectively targets mislocalized proteins and coordinates its activity with other ER functions, such as protein biogenesis, is unclear. Here, we uncover a link between INM proteome identity and membrane protein complex assembly in the remaining ER. We show that lone proteins and complex subunits failing to assemble in the ER access the INM for Asi-mediated degradation. Substrates are recognized by direct binding of Asi2 to their transmembrane domains for subsequent ubiquitination by Asi1/Asi3 and membrane extraction. Our data suggest a model in which spatial segregation of membrane protein complex assembly and quality control improves assembly efficiency and reduces the levels of orphan subunits.

In this issue of Molecular Cell, Hu et al. (2020) show that the cytosolic E3 ligase RNF126 reubiquitinates membrane proteins after their extraction from the membrane of the endoplasmic reticulum to target them for proteasomal degradation.

In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from the membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from the phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from Saccharomyces cerevisiae, we show that the ubiquitin ligase Doa10 (Teb-4/MARCH6 in animals) is a retrotranslocase that facilitates membrane protein extraction. A substrate's TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase. Luminal substrate segments cross the membrane in an unfolded state. Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action. Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction.

The structure of yeast Hrd1 in complex with Hrd3 shows that Hrd1 forms an aqueous cavity with a lateral seal within the endoplasmic reticulum membrane, shedding light on how misfolded proteins are transported out of the endoplasmic reticulum. Endoplasmic reticulum-associated protein degradation (ERAD) is an important internal quality-control pathway in cells. Misfolded proteins do not complete their maturation in the endoplasmic reticulum and are instead transported back to the cytoplasm for subsequent degradation. The activity of the transmembrane protein Hrd1 is essential for this process in yeast, but its function was unclear. Using cryoelectron microscopy, Tom Rapoport and colleagues solved the structure of Hrd1 in complex with Hrd3, its binding partner in the endoplasmic reticulum lumen. It emerges that, within this complex, some portions of Hrd1 form an aqueous cavity that extends from the cytoplasm almost to the endoplasmic reticulum lumen, whereas its other parts form a lateral seal within the reticulum membrane. This structure therefore indicates that Hrd1 forms a protein-conducting channel to facilitate the movement of rogue proteins out of the endoplasmic reticulum. Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome1,2,3,4—a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain5,6. Hrd1 is the crucial membrane component for retro-translocation7,8, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction—from the cytosol into or across membranes9,10,11. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.

Proteins that fail to fold correctly in the endoplasmic reticulum (ER) are retrotranslocated back into the cytosol where they become ubiquitinated and are eventually degraded by the proteasome. This process is called ER-associated protein degradation (ERAD). Recent research revealed that the ER-membrane protein Hrd1, an E3 ubiquitin ligase, forms a minimal system to translocate misfolded proteins across the membrane of the ER.

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins.

In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca2+ channels and couples Ca2+ influx to activation of calcineurin and of its substrate, the transcription factor NFAT. Here we show that an IAIIIT anchoring site in human AKAP79 binds the same surface of calcineurin as the PxIxIT recognition peptide of NFAT, albeit more strongly. A modest decrease in calcineurin-AKAP affinity due to an altered anchoring sequence is compatible with NFAT activation, whereas a further decrease impairs activation. Counterintuitively, increasing calcineurin-AKAP affinity increases recruitment of calcineurin to the scaffold but impairs NFAT activation; this is probably due to both slower release of active calcineurin from the scaffold and sequestration of active calcineurin by 'decoy' AKAP sites. We propose that calcineurin-AKAP79 scaffolding promotes NFAT signaling by balancing strong recruitment of calcineurin with its efficient release to communicate with NFAT.

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1.SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P2 binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P2-enriched plasma membrane.

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neuro-transmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca2+, and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.

Neurotransmission critically relies on synaptic vesicles fusing with the membrane of nerve cells to release their neurotransmitter content into the synaptic cleft, a process requiring the assembly of several members of the SNARE protein family. Stein et al. have now solved the X-ray crystallographic structure of an extended neuronal SNARE complex, which suggest that these proteins operate like nanomachines whose zippering all the way into the membranes triggers their fusion. Other SNAREs are likely to function as such robust and simple membrane fusion catalysts during most secretory or endocytosis events in eukaryotes. In neurotransmission, synaptic vesicles fuse with the membrane of nerve cells to release neurotransmitter content into the synaptic cleft. This process requires the assembly of several members of the SNARE protein family. Here, the X-ray structure of a neuronal SNARE complex is solved, providing insight into how these proteins assemble. Neurotransmission relies on synaptic vesicles fusing with the membrane of nerve cells to release their neurotransmitter content into the synaptic cleft, a process requiring the assembly of several members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family. SNAREs represent an evolutionarily conserved protein family that mediates membrane fusion in the secretory and endocytic pathways of eukaryotic cells1,2,3. On membrane contact, these proteins assemble in trans between the membranes as a bundle of four α-helices, with the energy released during assembly being thought to drive fusion4,5,6. However, it is unclear how the energy is transferred to the membranes and whether assembly is conformationally linked to fusion. Here, we report the X-ray structure of the neuronal SNARE complex, consisting of rat syntaxin 1A, SNAP-25 and synaptobrevin 2, with the carboxy-terminal linkers and transmembrane regions at 3.4 Å resolution. The structure shows that assembly proceeds beyond the already known core SNARE complex7, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region. Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most N-terminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.

Ca(2+)-dependent exocytosis of synaptic vesicles is mediated by the SNARE proteins synaptobrevin/VAMP, SNAP-25, and syntaxin. SNARE function is controlled by conserved regulatory proteins, including the complexins. In a study by Xue et al. in this issue of Neuron, contradictory data from Drosophila and mouse complexin mutants have been resolved, revealing a complex pattern of facilitatory and inhibitory domains.

Background: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing coreconstituted SNAREs as reaction partners, in fusion assays.Results: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca2+ does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin.Conclusions: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not-involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.