Two Coiled-Coil Domains of Chlamydia trachomatis IncA Affect Membrane Fusion Events during Infection

Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA’s fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.     

SNARE protein mimicry by an intracellular bacterium

Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.     

An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA

Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2.     

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.     

Luminescence detection of SNARE–SNARE interaction in Arabidopsis protoplasts

Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE–SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE–SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP–SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP–SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.     

Evidence for regulation of ER/Golgi SNARE complex formation by hsc70 chaperones

SNARE proteins control intracellular membrane fusion through formation of membrane-bridging helix bundles of amphipathic SNARE motifs. Repetitive cycles of membrane fusion likely involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, little is known about conformational regulation of SNAREs by other proteins. Here we demonstrate that hsc70 chaperones stimulate in vitro SNARE complex formation among the ER/Golgi SNAREs syntaxin 5, membrin, rbetl and sec22b, under conditions in which assembly is normally inhibited. Thus, molecular chaperones can render the SNARE motif more competent for assembly. Partially purified hsc70 fractions from brain cytosol had higher specific activities than fully purified hsc70, suggesting the involvement of unidentified cofactors. Using chemical crosslinking of cells followed by immunoprecipitation, we found that hsc70 was associated with ER/Golgi SNAREs in vivo. Consistent with a modulatory role for hsc70 in transport, we found that excess hsc70 specifically inhibited ER-to-Golgi transport in permeabilized cells.     

Structure and Dynamics of a Two‐Helix SNARE Complex in Live Cells

SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q_a‐, Q_b‐, Q_c‐ and R‐SNARE‐motif form a complex. The core complex represents a Q_aQ_bQ_cR‐SNARE‐motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)‐studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q_a), SNAP25 (Q_b/Q_c) and synaptobrevin 2 (R)]. We find that the Q_b‐SNARE‐motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q_b‐SNARE‐motif and depends on its position within the molecule. We conclude that a zippered Q_aQ_b‐SNARE complex represents a short‐lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.     

Multiple features within the syntaxin Sed5p mediate its Golgi localization

Protein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi—the Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells, the syntaxin SNARE Sed5p oversees membrane fusion events. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the steady‐state localization of Sed5p to the Golgi appears to be primarily conformation‐based relying on intra‐molecular associations between the Habc domain and SNARE‐motif while its tribasic COPI‐coatomer binding motif plays a role in intra‐Golgi retention.     

The syntaxins

Summary  

The SNARE hypothesis predicts that a family of SNAP receptors are localized to and function in diverse intracellular membrane compartments where membrane fusion processes take place. Syntaxins, the prototype family of SNARE proteins, have a carboxy-terminal tail-anchor and multiple coiled-coil domains. There are 15 members of the syntaxin family in the human genome and 7 syntaxin-like genes in the yeast Saccharomyces cerevisiae. In conjunction with other SNAREs and with the cytoplasmic NSF and SNAP proteins, syntaxins mediate vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway. They are crucial components that both drive and provide specificity to the myriad vesicular fusion processes that characterize the eukaryotic cell.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.     

SNARE function is not involved in early endosome docking

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific F_ab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.     

Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi

Background  

In eukaryotic cells, directional transport between different compartments of the endomembrane system is mediated by vesicles that bud from a donor organelle and then fuse with an acceptor organelle. A family of integral membrane proteins, termed soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, constitute the key machineries of these different membrane fusion events. Over the past 30 years, the yeast Saccharomyces cerevisiae has served as a powerful model organism for studying the organization of the secretory and endocytic pathways, and a few years ago, its entire set of SNAREs was compiled.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.     

Characteristics of SNARE proteins are defined by distinctive properties of SNARE motifs

BackgroundSyntaxin-1A and Sso1 are syntaxin family SNARE proteins engaged in synaptic vesicle fusion and yeast exocytosis. The syntaxin-1A SNARE motif can form a fusogenic SNARE complex with Sso1 partners. However, a chimera in which the SNARE motif in syntaxin-1A is introduced into Sso1 was not functional in yeast because the chimera is retained in the ER. Through the analysis of the transport defect of Sso1/syntaxin-1A chimeric SNAREs, we found that their SNARE motifs have distinctive properties.MethodsSso1, syntaxin-1A, and Sso1/syntaxin-1A chimeric SNAREs were expressed in yeast cells and their localization and interaction with other SNAREs are analyzed.ResultsSNARE proteins containing the syntaxin-1A SNARE motif exhibit a transport defect because they form a cis-SNARE complex in the ER. Ectopic SNARE complex formation can be prevented in syntaxin-1A by binding to a Sec1/Munc-18-like (SM) protein. In contrast, the SNARE motif of Sso1 does not form an ectopic SNARE complex. Additionally, we found that the SNARE motif in syntaxin-1A, but not that in Sso1, self-interacts, even when it is in the inactive form and bound to the SM protein.ConclusionsThe SNARE motif in syntaxin-1A, but not in Sso1, likely forms ectopic SNARE complex. Because of this property, the SM protein is necessary for syntaxin-1A to prevent its promiscuous assembly and to promote its export from the ER.General significanceProperties of SNARE motifs affect characteristics of SNARE proteins. The regulatory mechanisms of SNARE proteins are, in part, designed to handle such properties.     

Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H_abc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.     

LegC3, an Effector Protein from Legionella pneumophila,Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.     

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.     

The Habc domain and the SNARE core complex are connected by a highly flexible linker

Syntaxin 1a is a member of the SNARE superfamily of small, mostly membrane-bound proteins that mediate membrane fusion in all eukaryotic cells. Upon membrane fusion, syntaxin 1 forms a stable complex with its partner SNAREs. Syntaxin contains a C-terminal transmembrane domain, an adjacent SNARE motif that interacts with its partner SNAREs, and an N-terminal Habc domain. The Habc domain reversibly folds back upon the SNARE motif, resulting in a "closed" conformation that is stabilized by binding to the protein munc18. The SNARE motif and the Habc domain are separated by a linker region of about 40 amino acids. When syntaxin is complexed with munc18, the linker is structured and consists of a mix of turns and small alpha-helices. When syntaxin is complexed with its partner SNAREs, the Habc domain is dissociated, but the structure of the linker region is not known. Here we used site-directed spin labeling and EPR spectroscopy to determine the structure of the linker region of syntaxin in the SNARE complex. We found that the entire linker region of syntaxin is unstructured except for three residues at the N-terminal and six residues at the C-terminal boundary whereas the structures of the flanking regions in the Habc domain and the SNARE motif correspond to the high-resolution structures of the isolated fragments. We conclude that the linker region exhibits a high degree of conformational flexibility.