Multisubunit Tethering Complexes Research Articles

RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.

Oocyte-specific EXOC5 expression is required for mouse oogenesis and folliculogenesis.

EXOC5 is a crucial component of a large multi-subunit tethering complex, the exocyst complex, that is required for fusion of secretory vesicles with the plasma membrane. Exoc5 deleted mice die as early embryos. Therefore, to determine the role of EXOC5 in follicular and oocyte development, it was necessary to produce a conditional knockout (cKO), Zp3-Exoc5-cKO, in which Exoc5 was deleted only in oocytes. The first wave of folliculogenesis appeared histologically normal and progressed to the antral stage. However, after IVF with normal sperm, oocytes collected from the first wave (superovulated 21-day-old cKO mice) were shown to be developmentally incompetent. Adult follicular waves did not progress beyond the secondary follicle stage where they underwent apoptosis. Female cKO mice were infertile. Overall, these data suggest that the first wave of folliculogenesis is less sensitive to oocyte-specific loss of Exoc5, but the resulting gametes have reduced developmental competence. In contrast, subsequent waves of folliculogenesis require oocyte-specific Exoc5 for development past the preantral follicle stage. The Zp3-Exoc5-cKO mouse provides a model for disrupting folliculogenesis that also enables the separation between the first and subsequent waves of folliculogenesis.

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.

Structure of a membrane tethering complex incorporating multiple SNAREs.

Most membrane fusion reactions in eukaryotic cells are mediated by multisubunit 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-electron microscopy structure of the simplest known MTC, the 255-kDa Dsl1 complex of Saccharomyces cerevisiae, 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 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.

Highly efficient overexpression and purification of multisubunit tethering complexes in Saccharomyces cerevisiae

Vesicle trafficking is a fundamental cellular process that ensures proper material exchange between organelles in eukaryotic cells, and multisubunit tethering complexes (MTCs) are essential in this process. The heterohexameric homotypic fusion and protein sorting (HOPS) complex, which functions in the endolysosomal pathway, is a member of MTCs. Despite its critical role, the complex composition and low-expression level of HOPS have made its expression and purification extremely challenging. In this study, we present a highly efficient strategy for overexpressing and purifying HOPS from Saccharomyces cerevisiae. We achieved HOPS overexpression by integrating a strong promoter TEF1 before each subunit using the gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) system. The HOPS complex was subsequently purified using Staphylococcus aureus protein A (ProtA) affinity purification and size-exclusion chromatography, resulting in high purity and homogeneity. We obtained two-fold more HOPS using this method than that obtained using the commonly used GAL1 promoter-controlled HOPS overexpression. Negative staining electron microscopy analysis confirmed the correct assembly of HOPS. Notably, we also successfully purified two other MTCs, class C core vacuole/endosome tethering (CORVET) and Golgi-associated retrograde protein (GARP) using this approach. Our findings facilitate further in vitro biochemical characterization and functional studies of MTCs and provide a useful guide for the preparation of other heterogenic multisubunit complexes.

Role of GARP Vesicle Tethering Complex in Golgi Physiology.

The Golgi associated retrograde protein complex (GARP) is an evolutionarily conserved component of Golgi membrane trafficking machinery that belongs to the Complexes Associated with Tethering Containing Helical Rods (CATCHR) family. Like other multisubunit tethering complexes such as COG, Dsl1, and Exocyst, the GARP is believed to function by tethering and promoting fusion of the endosome-derived small trafficking intermediate. However, even twenty years after its discovery, the exact structure and the functions of GARP are still an enigma. Recent studies revealed novel roles for GARP in Golgi physiology and identified human patients with mutations in GARP subunits. In this review, we summarized our knowledge of the structure of the GARP complex, its protein partners, GARP functions related to Golgi physiology, as well as cellular defects associated with the dysfunction of GARP subunits.

The P4‐ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4‐ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4‐ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole‐specific subunit of TRAPPIII, to the N‐terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4‐ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4‐ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4‐ATPases in MTC regulation.

Comparative genomic analysis illustrates evolutionary dynamics of multisubunit tethering complexes across green algal diversity.

The chlorophyte algae are a dominant group of photosynthetic eukaryotes. Although many are photoautotrophs, there are also mixotrophs, heterotrophs, and even parasites. The physical characteristics of green algae are also highly diverse, varying greatly in size, shape, and habitat. Given this morphological and trophic diversity, we postulated that diversity may also exist in the protein components controlling intracellular movement of material by vesicular transport. One such set is the multisubunit tethering complexes (MTCs)-components regulating cargo delivery. As they span endomembrane organelles and are well-conserved across eukaryotes, MTCs should be a good proxy for assessing the evolutionary dynamics across the diversity of Chlorophyta. Our results reveal that while green algae carry a generally conserved and unduplicated complement of MTCs, some intriguing variation exists. Notably, we identified incomplete sets of TRAPPII, exocyst, and HOPS/CORVET components in all Mamiellophyceae, and what is more, not a single subunit of Dsl1 was found in Cymbomonas tetramitiformis. As the absence of Dsl1 has been correlated with having unusual peroxisomes, we searched for peroxisome biogenesis machinery, finding very few components in Cymbomonas, suggestive of peroxisome degeneration. Overall, we demonstrate conservation of MTCs across green algae, but with notable taxon-specific losses suggestive of unusual endomembrane systems.

Arabidopsis HOPS subunit VPS41 carries out plant-specific roles in vacuolar transport and vegetative growth.

The homotypic fusion and protein sorting (HOPS) complex is a conserved, multi-subunit tethering complex in eukaryotic cells. In yeast and mammalian cells, the HOPS subunit vacuolar protein sorting-associated protein 41 (VPS41) is recruited to late endosomes after Ras-related protein 7 (Rab7) activation and is essential for vacuole fusion. However, whether VPS41 plays conserved roles in plants is not clear. Here, we demonstrate that in the model plant Arabidopsis (Arabidopsis thaliana), VPS41 localizes to distinct condensates in root cells in addition to its reported localization at the tonoplast. The formation of condensates does not rely on the known upstream regulators but depends on VPS41 self-interaction and is essential for vegetative growth regulation. Genetic evidence indicates that VPS41 is required for both homotypic vacuole fusion and cargo sorting from the adaptor protein complex 3, Rab5, and Golgi-independent pathways but is dispensable for the Rab7 cargo inositol transporter 1. We also show that VPS41 has HOPS-independent functions in vacuolar transport. Taken together, our findings indicate that Arabidopsis VPS41 is a unique subunit of the HOPS complex that carries out plant-specific roles in both vacuolar transport and developmental regulation.

Getting Sugar Coating Right! The Role of the Golgi Trafficking Machinery in Glycosylation.

The Golgi is the central organelle of the secretory pathway and it houses the majority of the glycosylation machinery, which includes glycosylation enzymes and sugar transporters. Correct compartmentalization of the glycosylation machinery is achieved by retrograde vesicular trafficking as the secretory cargo moves forward by cisternal maturation. The vesicular trafficking machinery which includes vesicular coats, small GTPases, tethers and SNAREs, play a major role in coordinating the Golgi trafficking thereby achieving Golgi homeostasis. Glycosylation is a template-independent process, so its fidelity heavily relies on appropriate localization of the glycosylation machinery and Golgi homeostasis. Mutations in the glycosylation enzymes, sugar transporters, Golgi ion channels and several vesicle tethering factors cause congenital disorders of glycosylation (CDG) which encompass a group of multisystem disorders with varying severities. Here, we focus on the Golgi vesicle tethering and fusion machinery, namely, multisubunit tethering complexes and SNAREs and their role in Golgi trafficking and glycosylation. This review is a comprehensive summary of all the identified CDG causing mutations of the Golgi trafficking machinery in humans.

Recent Advances in Synergy Among SNARE Proteins and Multi-Subunit Tethering Complexes (MTCs) in Vesicle Trafficking and Plant Development

Plants comprise an expanded endomembrane system, and transport within the network requires well-organized and accurate vesicle transport. Tethering complexes facilitate the early, exact contact among donor and acceptor membranes, operate to bring vesicles into a closer proximity for trans-SNARE complex assembly docking; these are classified as either long coiled-coil proteins or multi-subunit tethering complexes (MTCs). Numerous MTCs that function at different membrane trafficking steps have been recognized, where they function as significant interfaces between SNARE proteins, Rabs, and phosphoinositides. SNARE proteins assemble into complexes that catalyze the fusion between a donor and a target membrane. Studies of the diverse SNARE proteins provided further valuable information about vacuole biogenesis and vacuolar trafficking pathways related to cell-type specificity, plant development, growth, and the plant developed a specific traffic route to overcome environmental stress. In conclusion, tethers' selective recruitment during membrane fusion is controlled via diverse small GTPases, such as those in the RAB family. The MTCs promote SNARE complex assembly by direct interactions of MTC subunits with Q-SNAREs. A subset of MTC subunits exploits structurally similar CATCHR domains to mediate inter-subunit interactions as well as SNARE protein interactions. MTCs are subdivided into CATCHR (complexes associated with tethering containing helical rods: Dsl1, COG, GARP, EARP, and exocyst) and non‐CATCHR (TRAPP I, II and III, HOPS and CORVET) complexes based on the structure of their subunits. This review summarized new information about SNARE proteins and tethering complexes, highlight new insights about their function, and discuss current debates and future perspectives.

The Exocyst Regulates Hydrolytic Enzyme Secretion at Hyphal Tips and Septa in the Banana Fusarium Wilt Fungus Fusarium odoratissimum.

Hyphal polarized growth in filamentous fungi requires tip-directed secretion, while additional evidence suggests that fungal exocytosis for the hydrolytic enzyme secretion can occur at other sites in hyphae, including the septum. In this study, we analyzed the role of the exocyst complex involved in the secretion in the banana wilt fungal pathogen Fusarium odoratissimum. All eight exocyst components in F. odoratissimum not only localized to the tips ahead of the Spitzenkörper in growing hyphae but also localized to the outer edges of septa in mature hyphae. To further analyze the exocyst in F. odoratissimum, we attempted single gene deletion for all the genes encoding the eight exocyst components and only succeeded in constructing the gene deletion mutants for exo70 and sec5; we suspect that the other 6 exocyst components are encoded by essential genes. Deletion of exo70 or sec5 led to defects in vegetative growth, conidiation, and pathogenicity in F. odoratissimum. Notably, the deletion of exo70 resulted in decreased activities for endoglucosidase, filter paper enzymes, and amylase, while the loss of sec5 only led to a slight reduction in amylase activity. Septum-localized α-amylase (AmyB) was identified as the marker for septum-directed secretion, and we found that Exo70 is essential for the localization of AmyB to septa. Meanwhile the loss of Sec5 did not affect AmyB localization to septa but led to a higher accumulation of AmyB in cytoplasm. This suggested that while Exo70 and Sec5 both take part in the septum-directed secretion, the two play different roles in this process. IMPORTANCE The exocyst complex is a multisubunit tethering complex (MTC) for secretory vesicles at the plasma membrane and contains eight subunits, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. While the exocyst complex is well defined in eukaryotes from yeast (Saccharomyces cerevisiae) to humans, the exocyst components in filamentous fungi show different localization patterns in the apical tips of hyphae, which suggests that filamentous fungi have evolved divergent strategies to regulate endomembrane trafficking. In this study, we demonstrated that the exocyst components in Fusarium odoratissimum are localized not only to the tips of growing hyphae but also to the outer edge of the septa in mature hyphae, suggesting that the exocyst complex plays a role in the regulation of septum-directed protein secretion in F. odoratissimum. We further found that Exo70 and Sec5 are required for the septum-directed secretion of α-amylase in F. odoratissimum but with different influences.

Homology and Modular Evolution of CATCHR at the Origin of the Eukaryotic Endomembrane System.

The membrane trafficking is an essential process of eukaryotic cells, as it manages vesicular trafficking toward different parts of the cell. In this process, membrane fusions between vesicles and target membranes are mediated by several factors, including the multisubunit tethering complexes. One type of multisubunit tethering complex, the complexes associated with tethering containing helical rods (CATCHR), encompasses the exocyst, COG, GARP, and DSL1 complexes. The CATCHR share similarities at sequence, structural, and protein-complex organization level although their actual relationship is still poorly understood. In this study, we have re-evaluated CATCHR at different levels, demonstrating that gene duplications followed by neofunctionalization, were key for their origin. Our results, reveals that there are specific homology relationships and parallelism within and between the CATCHR suggesting that most of these complexes are composed by modular tetramers of four different kinds of proteins, three of them having a clear common origin. The extension of CATCHR family occurred concomitantly with the protein family expansions of their molecular partners, such as small GTPases and SNAREs, among others, and likely providing functional specificity. Our results provide novel insights into the structural organization and mechanism of action of CATCHR, with implications for the evolution of the endomembrane system of eukaryotes and promoting CATCHR as ideal candidates to study the evolution of multiprotein complexes.

Activation of the Exocyst Tethering Complex for SNARE Complex Regulation and Membrane Fusion

A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high resolution structures of large, multisubunit tethering complexes (MTCs) that spatially and temporally control intracellular membrane fusion. Exocyst is a hetero-octameric protein complex, proposed to tether secretory vesicles at the plasma membrane, and to provide quality control of SNARE-mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence and single-particle cryo-EM, are providing critical insights into the structure and function of the exocyst. We are investigating how the yeast exocyst interacts with SNARE proteins to control SNARE complex formation and membrane fusion. Intriguingly, fully assembled exocyst interacts weakly with the individual SNAREs, SNARE complexes, and the SNARE regulator Sec1, despite previously observed robust SNARE binding with recombinant proteins. Using an auxin-inducible degradation system and mutant yeast strains, we purified exocyst subcomplexes and mutant complexes and showed that several have increased affinities for the different SNAREs. Negative stain EM was used to visualize the structure of an exocyst subcomplex, as well as the 3D structure of an activated mutant exocyst complex. Comparison of the negative stain images to the cryo-EM structure of fully assembled exocyst revealed that several subunits become more dynamic and accessible for SNARE interactions. We propose that exocyst needs to become activated and undergo one or more conformational changes, in order to efficiently interact with and regulate the SNARE proteins for membrane fusion.

The Small GTPase Arf6 Functions as a Membrane Tether in a Chemically-Defined Reconstitution System.

Arf-family small GTPases are essential protein components for membrane trafficking in all eukaryotic endomembrane systems, particularly during the formation of membrane-bound, coat protein complex-coated transport carriers. In addition to their roles in the transport carrier formation, a number of Arf-family GTPases have been reported to physically associate with coiled-coil tethering proteins and multisubunit tethering complexes, which are responsible for membrane tethering, a process of the initial contact between transport carriers and their target subcellular compartments. Nevertheless, whether and how indeed Arf GTPases are involved in the tethering process remain unclear. Here, using a chemically-defined reconstitution approach with purified proteins of two representative Arf isoforms in humans (Arf1, Arf6) and synthetic liposomes for model membranes, we discovered that Arf6 can function as a bona fide membrane tether, directly and physically linking two distinct lipid bilayers even in the absence of any other tethering factors, whereas Arf1 retained little potency to trigger membrane tethering under the current experimental conditions. Arf6-mediated membrane tethering reactions require trans-assembly of membrane-anchored Arf6 proteins and can be reversibly controlled by the membrane attachment and detachment cycle of Arf6. The intrinsic membrane tethering activity of Arf6 was further found to be significantly inhibited by the presence of membrane-anchored Arf1, suggesting that the tethering-competent Arf6-Arf6 assembly in trans can be prevented by the heterotypic Arf1-Arf6 association in a cis configuration. Taken together, these findings lead us to postulate that self-assemblies of Arf-family small GTPases on lipid bilayers contribute to driving and regulating the tethering events of intracellular membrane trafficking.

Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions.

Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

Golgi inCOGnito: From vesicle tethering to human disease

The Conserved Oligomeric Golgi (COG) complex, a multi-subunit vesicle tethering complex of the CATCHR (Complexes Associated with Tethering Containing Helical Rods) family, controls several aspects of cellular homeostasis by orchestrating retrograde vesicle traffic within the Golgi. The COG complex interacts with all key players regulating intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, and vesicular coats. In cells, COG deficiencies result in the accumulation of non-tethered COG-complex dependent (CCD) vesicles, dramatic morphological and functional abnormalities of the Golgi and endosomes, severe defects in N- and O- glycosylation, Golgi retrograde trafficking, sorting and protein secretion. In humans, COG mutations lead to severe multi-systemic diseases known as COG-Congenital Disorders of Glycosylation (COG-CDG). In this report, we review the current knowledge of the COG complex and analyze COG-related trafficking and glycosylation defects in COG-CDG patients.

Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1

Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-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.

Activation of the Exocyst Tethering Complex for SNARE Complex Regulation and Membrane Fusion

A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high resolution structures of large, multisubunit tethering complexes (MTCs) that spatially and temporally control intracellular membrane fusion. Exocyst is a hetero‐octameric protein complex, proposed to tether secretory vesicles at the plasma membrane, and to provide quality control of SNARE‐mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence and single‐particle cryo‐EM, are providing critical insights into the structure and function of the exocyst. We are investigating how the yeast exocyst interacts with SNARE proteins to control SNARE complex formation and membrane fusion. Intriguingly, fully assembled exocyst interacts weakly with the individual SNAREs, SNARE complexes, and the SNARE regulator Sec1, despite previously observed robust SNARE binding with recombinant proteins. Using an auxin‐inducible degradation system and mutant yeast strains, we purified exocyst subcomplexes and mutant complexes and showed that several have increased affinities for the different SNAREs. Negative stain EM was used to visualize the structure of an exocyst subcomplex, as well as the 3D structure of an activated mutant exocyst complex. Comparison of the negative stain images to the cryo‐EM structure of fully assembled exocyst revealed that several subunits become more dynamic and accessible for SNARE interactions. We propose that exocyst needs to become activated and undergo a conformational change, in order to efficiently interact with and regulate the SNAREs for membrane tethering and fusion.Support or Funding InformationWork in our laboratories is supported by US National Institutes of Health grants GM068803 (M.M.), GM054712 (P.B), F32GM123704 (D.L.) GM127673 (A.F.), DP2GM110772 (A.F.), A.F. is an American Asthma Foundation Scholar, an HHMI Faculty Scholar, and a Chan Zuckerberg Biohub Investigator. L.R.K. was supported by a graduate research fellowship from the NSF.