Most membrane fusion reactions in eukaryotic cells are mediated by membrane tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here, we present a cryo-EM structure of the simplest known MTC, the 255-kDa Dsl1 complex, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with remarkable and unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

The compartmentalization of eukaryotic cells is reliant on the fidelity of vesicle-mediated intracellular transport. Vesicles deliver their cargo via membrane fusion, a process requiring membrane tethers, Sec1/Munc18 (SM) proteins, and SNAREs. These components function in concert to ensure that membrane fusion is efficient and accurate, but the mechanisms underlying their cooperative action are still in many respects mysterious. In this brief review, we highlight recent progress toward a more integrative understanding of the vesicle fusion machinery. We focus particular attention on cryo-electron microscopy structures of intact multisubunit tethers in complex with SNAREs or SM proteins, as well as a structure of an SM protein bound to multiple SNAREs. The insights gained from this work emphasize the advantages of studying the fusion machinery intact and in context.

SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM-Qa-SNARE complex, Vps45-Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45-Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery.

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.

Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

Coat protein I (COPI)-coated vesicles mediate retrograde transport from the Golgi to the endoplasmic reticulum (ER), as well as transport within the Golgi. Major progress has been made in defining the structure of COPI coats, in vitro and in vivo, at resolutions as high as 9 Å. Nevertheless, important questions remain unanswered, including what specific interactions stabilize COPI coats, how COPI vesicles recognize their target membranes, and how coat disassembly is coordinated with vesicle fusion and cargo delivery. Here, we use X-ray crystallography to identify a conserved site on the COPI subunit α-COP that binds to flexible, acidic sequences containing a single tryptophan residue. One such sequence, found within α-COP itself, mediates α-COP homo-oligomerization. Another such sequence is contained within the lasso of the ER-resident Dsl1 complex, where it helps mediate the tethering of Golgi-derived COPI vesicles at the ER membrane. Together, our findings suggest that α-COP homo-oligomerization plays a key role in COPI coat stability, with potential implications for the coordination of vesicle tethering, uncoating, and fusion.

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors' kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.

The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.

Fusion of intracellular transport vesicles requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18-family (SM) proteins. Membrane-bridging SNARE complexes are critical for fusion, but their spontaneous assembly is inefficient and may require SM proteins in vivo. We report x-ray structures of Vps33, the SM subunit of the yeast homotypic fusion and vacuole protein-sorting (HOPS) complex, bound to two individual SNAREs. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent complex assembly. Vps33 and potentially other SM proteins could thus act as templates for generating partially zipped SNARE assembly intermediates. HOPS was essential to mediate SNARE complex assembly at physiological SNARE concentrations. Thus, Vps33 appears to catalyze SNARE complex assembly through specific SNARE motif recognition.

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1-4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG's eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5-Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5-Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation.

The peripheral endoplasmic reticulum (ER) network is dynamically maintained by homotypic (ER-ER) fusion. In Saccharomyces cerevisiae, the dynamin-like GTPase Sey1p can mediate ER-ER fusion, but sey1Δ cells have no growth defect and only slightly perturbed ER structure. Recent work suggested that ER-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate a Sey1p-independent ER-ER fusion pathway. However, an alternative explanation--that the observed phenotypes arose from perturbed vesicle trafficking--could not be ruled out. In this study, we used candidate and synthetic genetic array (SGA) approaches to more fully characterize SNARE-mediated ER-ER fusion. We found that Dsl1 complex mutations in sey1Δ cells cause strong synthetic growth and ER structure defects and delayed ER-ER fusion in vivo, additionally implicating the Dsl1 complex in SNARE-mediated ER-ER fusion. In contrast, cytosolic coat protein I (COPI) vesicle coat mutations in sey1Δ cells caused no synthetic defects, excluding perturbed retrograde trafficking as a cause for the previously observed synthetic defects. Finally, deleting the reticulons that help maintain ER architecture in cells disrupted for both ER-ER fusion pathways caused almost complete inviability. We conclude that the ER SNAREs and the Dsl1 complex directly mediate Sey1p-independent ER-ER fusion and that, in the absence of both pathways, cell viability depends upon membrane curvature-promoting reticulons.

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the "K loop," which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ∼60 Å, twice the ∼30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multidomain proteins.

The assembly of COPI into a cage-like lattice sculpts membrane vesicles that transport cargo from the Golgi apparatus. Now, Lee and Goldberg (2010) present X-ray crystal structures of COPI suggesting that these coats combine selected features of two other archetypal coats, clathrin and COPII.

Cell biologists have long been interested in understanding the machinery that mediates movement of proteins and lipids between intracellular compartments. Much of this traffic is accomplished by vesicles (or other membranous carriers) that bud from one compartment and fuse with another. Given the pivotal roles that large protein complexes play in vesicular trafficking, many recent advances have relied on the combined use of X-ray crystallography and electron microscopy. Here, we discuss integrated structural studies of proteins whose assembly shapes membranes into vesicles and tubules, before turning to the so-called tethering factors that appear to orchestrate vesicle docking and fusion.

Multisubunit tethering complexes of the CATCHR (complexes associated with tethering containing helical rods) family are proposed to mediate the initial contact between an intracellular trafficking vesicle and its membrane target. To begin elucidating the molecular architecture of one well-studied example, the conserved oligomeric Golgi (COG) complex, we reconstituted its essential subunits (Cog1, Cog2, Cog3 and Cog4) and used single-particle electron microscopy to reveal a y-shaped structure with three flexible, highly extended legs. Labeling experiments established that the N termini of all four subunits interact along the proximal segment of one leg, whereas three of the four C termini are located at the tips of the legs. Our results suggest that the central region of the Cog1-Cog2-Cog3-Cog4 complex, as well as the distal regions of at least two legs, all participate in interactions with other components of the intracellular trafficking machinery.

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release > or =100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.

Vesicle trafficking requires membrane fusion, mediated by SNARE proteins, and upstream events that probably include "tethering," an initial long-range attachment between a vesicle and its target organelle. Among the factors proposed to mediate tethering are a set of multisubunit tethering complexes (MTCs). The Dsl1 complex, with only three subunits, is the simplest known MTC and is essential for the retrograde traffic of COPI-coated vesicles from the Golgi to the ER. To elucidate structural principles underlying MTC function, we have determined the structure of the Dsl1 complex, revealing a tower containing at its base the binding sites for two ER SNAREs and at its tip a flexible lasso for capturing vesicles. The Dsl1 complex binds to individual SNAREs via their N-terminal regulatory domains and also to assembled SNARE complexes; moreover, it is capable of accelerating SNARE complex assembly. Our results suggest that even the simplest MTC may be capable of orchestrating vesicle capture, uncoating, and fusion.

The proper glycosylation of proteins trafficking through the Golgi apparatus depends upon the conserved oligomeric Golgi (COG) complex. Defects in COG can cause fatal congenital disorders of glycosylation (CDGs) in humans. The recent discovery of a form of CDG, caused in part by a COG4 missense mutation changing Arg 729 to Trp, prompted us to determine the 1.9 A crystal structure of a Cog4 C-terminal fragment. Arg 729 is found to occupy a key position at the center of a salt bridge network, thereby stabilizing Cog4's small C-terminal domain. Studies in HeLa cells reveal that this C-terminal domain, while not needed for the incorporation of Cog4 into COG complexes, is essential for the proper glycosylation of cell surface proteins. We also find that Cog4 bears a strong structural resemblance to exocyst and Dsl1p complex subunits. These complexes and others have been proposed to function by mediating the initial tethering between transport vesicles and their membrane targets; the emerging structural similarities provide strong evidence of a common evolutionary origin and may reflect shared mechanisms of action.

Multisubunit tethering complexes are essential for intracellular trafficking and have been proposed to mediate the initial interaction between vesicles and the membranes with which they fuse. Here we report initial structural characterization of the Dsl1p complex, whose three subunits are essential for trafficking from the Golgi apparatus to the endoplasmic reticulum (ER). Crystal structures reveal that two of the three subunits, Tip20p and Dsl1p, resemble known subunits of the exocyst complex, establishing a structural connection among several multisubunit tethering complexes and implying that many of their subunits are derived from a common progenitor. We show, moreover, that Tip20p and Dsl1p interact directly via N-terminal alpha-helices. Finally, we establish that different Dsl1p complex subunits bind independently to different ER SNARE proteins. Our results map out two alternative protein-interaction networks capable of tethering COPI-coated vesicles, via the Dsl1p complex, to ER membranes.

Anterograde transport in the early secretory pathway is mediated by COPII-coated vesicles. Stagg et al. (2008) have now visualized the double-layered COPII coat using electron cryomicroscopy, providing insight into how coats are assembled to accommodate cargo of different sizes.

The conserved oligomeric Golgi (COG) complex is strongly implicated in retrograde vesicular trafficking within the Golgi apparatus. Although its mechanism of action is poorly understood, it has been proposed to function by mediating the initial physical contact between transport vesicles and their membrane targets. An analogous role in tethering vesicles has been suggested for at least six additional large multisubunit complexes, including the exocyst, a complex essential for trafficking to the plasma membrane. Here we report the solution structure of a large portion of yeast Cog2p, one of eight subunits composing the COG complex. The structure reveals a six-helix bundle with few conserved surface features but a general resemblance to recently determined crystal structures of four different exocyst subunits. This finding provides the first structural evidence that COG, like the exocyst and potentially other tethering complexes, is constructed from helical bundles. These structures may represent platforms for interaction with other trafficking proteins including SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) and Rabs.

Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

The conserved oligomeric Golgi (COG) complex is essential for establishing and/or maintaining the structure and function of the Golgi apparatus. The Golgi apparatus, in turn, has a central role in protein sorting and glycosylation within the eukaryotic secretory pathway. As a consequence, COG mutations can give rise to human genetic diseases known as congenital disorders of glycosylation. We review recent results from studies of yeast, worm, fly and mammalian COG that provide evidence that COG might function in retrograde vesicular trafficking within the Golgi apparatus. This hypothesis explains the impact of COG mutations by postulating that they impair the retrograde flow of resident Golgi proteins needed to maintain normal Golgi structure and function.

The extracellular signaling molecule autoinducer-2 (AI-2) mediates quorum-sensing communication in diverse bacterial species. In marine vibrios, binding of AI-2 to the periplasmic receptor LuxP modulates the activity of the inner membrane sensor kinase LuxQ, transducing the AI-2 information into the cytoplasm. Here, we show that Vibrio harveyi LuxP associates with LuxQ in both the presence and absence of AI-2. The 1.9 A X-ray crystal structure of apoLuxP, complexed with the periplasmic domain of LuxQ, reveals that the latter contains two tandem Per/ARNT/Simple-minded (PAS) folds. Thus, although many prokaryotic PAS folds themselves bind ligands, the LuxQ periplasmic PAS folds instead bind LuxP, monitoring its AI-2 occupancy. Mutations that disrupt the apoLuxP:LuxQ interface sensitize V. harveyi to AI-2, implying that AI-2 binding causes the replacement of one set of LuxP:LuxQ contacts with another. These conformational changes switch LuxQ between two opposing enzymatic activities, each of which conveys information to the cytoplasm about the cell density of the surrounding environment.

The conserved oligomeric Golgi (COG) complex is thought to function in intra-Golgi retrograde trafficking mediated by coat protein I vesicles, a pathway essential for the proper structure and function of the Golgi apparatus. Previous work suggested that COG might act as a tethering factor to mediate the initial attachment between coat protein I vesicles and Golgi membranes. Here, we present extensive in vitro co-translation and immunoprecipitation experiments leading to a new model for the overall architecture of the mammalian COG complex. The eight COG subunits (Cog1-8) are found to form two heterotrimeric subassemblies (Cog2/3/4 and Cog5/6/7) linked by a heterodimer composed of the remaining subunits (Cog1/8). This model is in excellent agreement with in vivo data presented in an accompanying paper (Oka, T., Vasile, E., Penman, M., Novina, C. D., Dykxhoorn, D. M., Ungar, D., Hughson, F. M., and Krieger, M. (2005) J. Biol. Chem. 280, 32736-32745).

Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi, is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium. We find that LsrB binds a chemically distinct form of the AI-2 signal, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling.

Cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development. Unlike other autoinducers, which are specific to a particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species. AI-2 has been proposed to serve as a 'universal' signal for inter-species communication. The chemical identity of AI-2 has, however, proved elusive. Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with autoinducer. The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized autoinducers. Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2. Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.

SNAP receptor (SNARE) proteins function in intracellular trafficking by forming complexes that bridge vesicle and target membranes prior to fusion. Biochemical studies indicate that the entry of certain SNARE proteins into complexes is inhibited by intramolecular interactions that generate a closed conformation. For example, an essential N-terminal regulatory domain of the yeast plasma membrane SNARE Sso1p sequesters the C-terminal SNARE motif and prevents it from binding to its assembly partners Sec9p and Sncp. Here, we introduce mutations into Sso1p that cause it to remain constitutively open. These open mutants can functionally substitute for wild-type Sso1p protein in vivo, demonstrating that inhibition of SNARE assembly is not the essential function of the N-terminal regulatory domain. Furthermore, the open mutants suppress sec9--4, a mutation that causes a severe defect in SNARE assembly. Elevated levels of SNARE complexes are observed in cells expressing the open mutants. In the presence of sufficient Sec9p, these complexes accumulate to levels that cause severe growth defects. Similarly, overexpression of the open mutants in yeast carrying mutations in the SNARE disassembly machinery impairs growth. Our findings indicate that elevated levels of SNARE complexes can be toxic and that these levels are normally controlled by the SNARE disassembly machinery, by the limited availability of Sec9p, and by the closed conformation of Sso1p.