Multisubunit Tethering Complexes Research Articles

Characterization of phospholipid binding by the exocyst complex using lipid nanodiscs

Proper regulation of exocytosis is crucial for cell function. Post‐Golgi vesicles require precise localization and temporal specification to fuse at correct sites on the plasma membrane to secrete their cargo 1. Secretion is essential for cell survival and is involved in intercellular communication and cell migration 2. Multisubunit tethering complexes (MTCs) are protein assemblies that coordinate interactions required for membrane fusion. Exocyst is a hetero‐octameric MTC responsible for targeting secretory vesicles to the plasma membrane by recognizing SNAREs, small GTPases, and phospholipid 2. Two exocyst subunits, Sec3 and Exo70, have been shown to bind phosphatidylinositol 4,5 biphosphate (PI(4,5)P2). Sec3 contains an N‐terminal Pleckstrin homology (PH) domain and there is a positively charged patch in the C‐terminus of Exo70 3, 4. These two domains mediate interactions with PI(4,5)P2. However, these interactions have only been shown using recombinantly purified individual subunits or domains. Combining exocyst purified from cryo‐milled yeast lysate with lipid nanodiscs, we are able to study binding of the wildtype and mutant octameric complex to various phospholipids. This has allowed us to characterize differential contributions of Sec3 and Exo70 to PI(4,5)P2 binding. Further, nanodiscs allow us to investigate interactions of the exocyst complex with a wide range of phospholipids, demonstrating the specificity of exocyst‐PI(4,5)P2 binding. This research is crucial to understanding the mechanism by which exocytic machinery is targeted to sites of exocytosis.

Exocyst Tethering Complex Regulation of SNARE Proteins 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 fusion.

Defective Quiescent Center/AtTRS85 Encoding a TRAPPIII-specific Subunit Required for the Trans-golgi Network/Early Endosome Integrity is Essential for the Proper Root Development in Arabidopsis

Tethering factors mediate the initial contact between donor and acceptor membranes. Transport protein particle (TRAPP)III, a multisubunit tethering complex, controls both secretory pathway and autophagy in yeast. However, the roles of TRAPPIII is poorly understood in plants. We have isolated a recessive mutant displaying retarded root growth, defective root hair elongation, and compromised organization of quiescent center by introducing UAS tag in Arabidopsis thaliana and named defective quiescent center (dqc)-1. The tag localized in the 25th exon of At5g16280 encoding AtTRS85 specifying the TRAPPIII complex. Three additional T-DNA insertion mutants of dqc/attrs85 exhibited the defective gravitropic responses in addition to the weak dqc-1-like phenotypes. In dqc-2, the auxin-responsive DR5 expression in the columella was extended and an auxin efflux carrier PIN-FORMED1-GFP abnormally aggregated in the vacuole-like subcellular compartments. In the root epidermis of dqc-2, an early endosome (EE) marker was dispersed in the cytoplasm, a trans-Golgi network (TGN) marker localized in tonoplast, and a vacuole marker localized in the fragmented tonoplast. Furthermore, the endocytosis of FM4-64 in dqc-2 was compromised as evidenced by the decrease in the EE punctum formation, the rapid accumlation into the tonoplast, and the infrequent brefeldin A body formation. Together, DQC/AtTRS85 is essential for the integrity of TGN/EE and the vacuole fusion.

Maintaining order: COG complex controls Golgi trafficking, processing, and sorting.

The conserved oligomeric Golgi (COG) complex, a multisubunit tethering complex of the CATCHR (complexes associated with tethering containing helical rods) family, controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle targeting within the Golgi. In humans, COG defects lead to severe multisystemic diseases known as COG‐congenital disorders of glycosylation (COG‐CDG). The COG complex both physically and functionally interacts with all classes of molecules maintaining intra‐Golgi trafficking, namely SNAREs, SNARE‐interacting proteins, Rabs, coiled‐coil tethers, and vesicular coats. Here, we review our current knowledge of COG‐related trafficking and glycosylation defects in humans and model organisms, and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting.

Defects in COG-Mediated Golgi Trafficking Alter Endo-Lysosomal System in Human Cells.

The conserved oligomeric complex (COG) is a multi-subunit vesicle tethering complex that functions in retrograde trafficking at the Golgi. We have previously demonstrated that the formation of enlarged endo-lysosomal structures (EELSs) is one of the major glycosylation-independent phenotypes of cells depleted for individual COG complex subunits. Here, we characterize the EELSs in HEK293T cells using microscopy and biochemical approaches. Our analysis revealed that the EELSs are highly acidic and that vATPase-dependent acidification is essential for the maintenance of this enlarged compartment. The EELSs are accessible to both trans-Golgi enzymes and endocytic cargo. Moreover, the EELSs specifically accumulate endolysosomal proteins Lamp2, CD63, Rab7, Rab9, Rab39, Vamp7, and STX8 on their surface. The EELSs are distinct from lysosomes and do not accumulate active Cathepsin B. Retention using selective hooks (RUSH) experiments revealed that biosynthetic cargo mCherry-Lamp1 reaches the EELSs much faster as compared to both receptor-mediated and soluble endocytic cargo, indicating TGN origin of the EELSs. In support to this hypothesis, EELSs are enriched with TGN specific lipid PI4P. Additionally, analysis of COG4/VPS54 double KO cells revealed that the activity of the GARP tethering complex is necessary for EELSs’ accumulation, indicating that protein mistargeting and the imbalance of Golgi-endosome membrane flow leads to the formation of EELSs in COG-deficient cells. The EELSs are likely to serve as a degradative storage hybrid organelle for mistargeted Golgi enzymes and underglycosylated glycoconjugates. To our knowledge this is the first report of the formation of an enlarged hybrid endosomal compartment in a response to malfunction of the intra-Golgi trafficking machinery.

Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt.

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. We have previously identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells (Lőrincz et al., 2016a). Here, we show that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Lt and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.

Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering

Membrane tethering is a highly regulated event occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g. Rab/lipid molar ratios of 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab/lipid ratio of 1:3,000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.

Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion.

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeastSec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.

Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. Class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) are two such complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named “CHEVI”), perhaps analogous in configuration to CORVET and HOPS. We utilized the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting that these proteins have distinct sub-cellular localizations. Consistent with previous reports, we observed that VPS33A was co-localized with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localized with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localize with VPS33B. Instead, we identified that VPS33B interacts directly with CCDC22, a member of the CCC complex. CCDC22 does not co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates, suggesting that CCDC22 interacts transiently with VPS33B/VIPAR rather than forming a stable complex with these proteins in cells. We also observed that the protein complex containing VPS33B and VIPAR is considerably smaller than CORVET/HOPS, suggesting that the CHEVI complex comprises just VPS33B and VIPAR.

Membrane detachment is not essential for COG complex function.

COG is a multisubunit vesicle tethering complex in the Golgi. We demonstrate that both COG subcomplexes can be permanently attached to Golgi membranes and that major COG functions do not require cycling between the membrane and cytosol.

Detailed Analysis of the Interaction of Yeast COG Complex.

The Golgi apparatus is a central station for protein trafficking in eukaryotic cells. A widely accepted model of protein transport within the Golgi apparatus is cisternal maturation. Each cisterna has specific resident proteins, which are thought to be maintained by COPI-mediated transport. However, the mechanisms underlying specific sorting of these Golgi-resident proteins remain elusive. To obtain a clue to understand the selective sorting of vesicles between the Golgi cisterenae, we investigated the molecular arrangements of the conserved oligomeric Golgi (COG) subunits in yeast cells. Mutations in COG subunits cause defects in Golgi trafficking and glycosylation of proteins and are causative of Congenital Disorders of Glycosylation (CDG) in humans. Interactions among COG subunits in cytosolic and membrane fractions were investigated by co-immunoprecipitation. Cytosolic COG subunits existed as octamers, whereas membrane-associated COG subunits formed a variety of subcomplexes. Relocation of individual COG subunits to mitochondria resulted in recruitment of only a limited number of other COG subunits to mitochondria. These results indicate that COG proteins function in the forms of a variety of subcomplexes and suggest that the COG complex does not comprise stable tethering without other interactors.Key words: The Golgi apparatus, COG complex, yeast, membrane trafficking, multi-subunit tethering complex.

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.

Reconstitution of membrane tethering mediated by Rab-family small GTPases.

Membrane tethering is one of the most critical steps to determine the spatiotemporal specificity of membrane trafficking, which is the process to selectively transport proteins, lipids, and other biological molecules to the appropriate locations in eukaryotic cells, such as subcellular organelles, the plasma membrane, and the extracellular space. Based on genetic, cell biological, biochemical, and structural studies, Rab-family small GTPases and a number of Rab-interacting proteins (termed Rab effectors), including coiled-coil tethering proteins and multisubunit tethering complexes, have been proposed to be key protein components for membrane tethering. Nevertheless, indeed whether and how Rab GTPases and their specific Rab effectors directly act upon and catalyze membrane tethering still remains enigmatic. By chemically defined reconstitution of membrane tethering from purified Rab-family GTPase proteins and synthetic liposomal membranes, recent studies have revealed the intrinsic potency of Rab-family GTPases to physically and specifically tether two distinct lipid bilayers of liposomal membranes. Experimental evidence from these reconstitution studies support the novel working model in which Rab-family small GTPases act as a bona fide membrane tether for mediating membrane tethering events in eukaryotic membrane trafficking.

Human Rab small GTPase– and class V myosin–mediated membrane tethering in a chemically defined reconstitution system

Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions.

VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

The FgVps39-FgVam7-FgSso1 Complex Mediates Vesicle Trafficking and Is Important for the Development and Virulence of Fusarium graminearum.

Vesicle trafficking is an important event in eukaryotic organisms. Many proteins and lipids transported between different organelles or compartments are essential for survival. These processes are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Rab-GTPases, and multisubunit tethering complexes such as class C core vacuole or endosome tethering and homotypic fusion or vacuole protein sorting (HOPS). Our previous study has demonstrated that FgVam7, which encodes a SNARE protein involving in vesicle trafficking, plays crucial roles in growth, asexual or sexual development, deoxynivalenol production, and pathogenicity in Fusarium graminearum. Here, the affinity purification approach was used to identify FgVam7-interacting proteins to explore its regulatory mechanisms during vesicle trafficking. The orthologs of yeast Vps39, a HOPS tethering complex subunit, and Sso1, a SNARE protein localized to the vacuole or endosome, were identified and selected for further characterization. In yeast two-hybrid and glutathione-S-transferase pull-down assays, FgVam7, FgVps39, and FgSso1 interacted with each other as a complex. The ∆Fgvps39 mutant generated by targeted deletion was significantly reduced in vegetative growth and asexual development. It failed to produce sexual spores and was defective in plant infection and deoxynivalenol production. Further cellular localization and cytological examinations suggested that FgVps39 is involved in vesicle trafficking from early or late endosomes to vacuoles in F. graminearum. Additionally, the ∆Fgvps39 mutant was defective in vacuole morphology and autophagy, and it was delayed in endocytosis. Our results demonstrate that FgVam7 interacts with FgVps39 and FgSso1 to form a unique complex, which is involved in vesicle trafficking and modulating the proper development of infection-related morphogenesis in F. graminearum.

Know when to Hold ‘Em, Know when to Fold ‘Em; SM Proteins as Templates for SNARE Assembly

A major focus of our research is the machinery that guides the fusion of vesicles during intracellular transport. These vesicles deliver their cargo via membrane fusion reactions executed by membrane-bridging SNARE complexes. The formation of SNARE complexes generally requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. Consequently, we hypothesize that SNARE complex assembly reactions in the cell are orchestrated by a set of ‘topologically aware’ chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with - and sometimes as integral subunits of - the MTCs. Therefore, the goal of our work is to achieve an improved structural and mechanistic understanding of MTC and SM function in the assembly of fusogenic SNARE complexes. My presentation will focus on the SM proteins, and on our X-ray crystallographic and biochemical efforts to understand how they catalyze four-helix bundle formation.

Conserved Oligomeric Golgi and Neuronal Vesicular Trafficking.

The conserved oligomeric Golgi (COG) complex is an evolutionary conserved multi-subunit vesicle tethering complex essential for the majority of Golgi apparatus functions: protein and lipid glycosylation and protein sorting. COG is present in neuronal cells, but the repertoire of COG function in different Golgi-like compartments is an enigma. Defects in COG subunits cause alteration of Golgi morphology, protein trafficking, and glycosylation resulting in human congenital disorders of glycosylation (CDG) type II. In this review we summarize and critically analyze recent advances in the function of Golgi and Golgi-like compartments in neuronal cells and functions and dysfunctions of the COG complex and its partner proteins.

CATCHR, HOPS and CORVET tethering complexes share a similar architecture.

We show that the Saccharomyces cerevisiae GARP complex and the Cog1-4 subcomplex of the COG complex, both members of the complexes associated with tethering containing helical rods (CATCHR) family of multisubunit tethering complexes, share the same subunit organization. We also show that the HOPS complex, a tethering complex acting in the endolysosomal pathway, shares a similar architecture, suggesting that multisubunit tethering complexes use related structural frameworks.

Coat/Tether Interactions-Exception or Rule?

Coat complexes are important for cargo selection and vesicle formation. Recent evidence suggests that they may also be involved in vesicle targeting. Tethering factors, which form an initial bridge between vesicles and the target membrane, may bind to coat complexes. In this review, we ask whether these coat/tether interactions share some common mechanisms, or whether they are special adaptations to the needs of very specific transport steps. We compare recent findings in two multisubunit tethering complexes, the Dsl1 complex and the HOPS complex, and put them into context with the TRAPP I complex as a prominent example for coat/tether interactions. We explore where coat/tether interactions are found, compare their function and structure, and comment on a possible evolution from a common ancestor of coats and tethers.