N-ethylmaleimide-sensitive Factor Attachment Protein Receptor Research Articles

Requirements of Qa-SNARE LjSYP132s for Nodulation and Seed Development in Lotus japonicus

SNAREs (soluble N-ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti-inoculated roots of mutants, nfr1, castor and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds.

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ–activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane–localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane–localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D–overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ–activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Doc2b Protects β-Cells Against Inflammatory Damage and Enhances Function.

Loss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass.

A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly.

Whereas SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the three integrally anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a transmembrane anchor) or their SNARE heptad-repeat domains. Fusion required a transmembrane-anchored R-SNARE on one membrane and an anchored Q-SNARE on the other. The N-domain of the Qb-SNARE was completely dispensable for fusion. Whereas fusion can be promoted by very high concentrations of the Qa-SNARE heptad-repeat domain alone, at physiological concentrations the Qa-SNARE heptad-repeat domain alone has almost no fusion activity. The 181-198 region of Qa, immediately upstream of the SNARE heptad-repeat domain, is required for normal fusion activity with HOPS. This region is needed for normal SNARE complex assembly.

Identification of syntaxin 4 as an essential factor for the hepatitis C virus life cycle

Although there is evidence that multivesicular bodies (MVBs) are involved in the release of hepatitis C virus (HCV), many aspects of HCV release are still not fully understood. The amount of α-taxilin that prevents SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) complex formation by binding to free syntaxin 4 is reduced in HCV-positive cells. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 which mediates vesicles fusion is involved in the HCV life cycle.HCV-positive cells possess an increased amount of syntaxin 4 protein, although the amount of syntaxin 4-specific transcripts is decreased in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes. In HCV-positive cells a significant longer half-life of syntaxin 4 was found that overcompensates for the decreased expression and leads to the elevated level of syntaxin 4.Overexpression of syntaxin 4 reduces the intracellular amount of infectious viral particles by facilitating viral release, while silencing of syntaxin 4 expression using specific siRNAs inhibits the release of HCV particles and so leads to an increase in the intracellular amount of infectious viral particles. This indicates that HCV uses a SNARE-dependent pathway for viral release. Confocal immunofluorescence microscopy revealed a colocalization of syntaxin 4 with a MVB-specific marker, exosomes and HCV core, which suggests a fraction of syntaxin 4 is associated with exosomes loaded with HCV. Altogether, it is assumed that syntaxin 4 is a novel essential cellular factor for the release of HCV.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells.

Multiple ER-Golgi SNARE transmembrane domains are dispensable for trafficking but required for SNARE recycling.

The formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between opposing membranes is an essential prerequisite for fusion between vesicles and their target compartments. The composition and length of a SNARE's transmembrane domain (TMD) is also an indicator for their steady-state distribution in cells. The evolutionary conservation of the SNARE TMD, together with the strict requirement of this feature for membrane fusion in biochemical studies, implies that the TMD represents an essential protein module. Paradoxically, we find that for several essential ER- and Golgi-localized SNAREs, a TMD is unnecessary. Moreover, in the absence of a covalent membrane tether, such SNAREs can still support ER-Golgi vesicle transport and recapitulate established genetic interactions. Transport anomalies appear to be restricted to retrograde trafficking, but these defects are overcome by the attachment of a C-terminal lipid anchor to the SNARE. We conclude that the TMD functions principally to support the recycling of Qb-, Qc-, and R-SNAREs and, in so doing, retrograde transport.

Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet β-Cells.

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7’s precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus–short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca2+-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se.

Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking.

The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown. We investigated two such proteins, epsinR and gadkin, using the knocksideways method, which rapidly depletes proteins from the available pool by trapping them onto mitochondria. Although epsinR is known to be an N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-specific adaptor, the epsinR knocksideways blocked the production of the entire population of intracellular clathrin-coated vesicles (CCVs), suggesting a more global function. Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins. Both sides of the vesicle are densely covered, indicating that CCVs sort their cargo by molecular crowding. Trapping of gadkin onto mitochondria also blocked the production of intracellular CCVs but by a different mechanism: vesicles became cross-linked to mitochondria and pulled out toward the cell periphery. Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches.

Protein sorting to intracellular compartments.

The organization of eukaryotic cells depends on correct targeting of proteins and lipids to the organelles at which they function, but how organelle identity is maintained in the face of a constant flux of material is a major problem for the cell. New mechanisms addressing this question were revealed at the Minisymposium on “Membrane Traffic: Dynamic and Regulation.” Starting with the endoplasmic reticulum (ER), Silvere Pagant (Miller lab) showed that some plasma membrane (PM) proteins require multiple signals to drive capture into COPII vesicles. One signal comes from the cargo protein itself, and the second comes signal from a cargo receptor; both signals simultaneously engage the cargo adaptor for the COPII coat, Sec24. Prasanna Satpute-Krishnan (Lippincott-Schwartz and Hegde labs), recipient of the 2014 Merton Bernfield Award, presented her work on a novel quality-control mechanism for glycosylphosphatidylinositol (GPI)-anchored proteins called RESET (for rapid ER stress-induced export). ER stress causes receptor-mediated exit of GPI-anchored proteins from the ER through the secretory pathway to the PM, from which they are endocytosed and degraded in lysosomes. Only misfolded GPI-anchored proteins bind tightly to the cargo receptor, Tmp21, providing a new paradigm in ER stress clearance. Moving from the ER to the Golgi, Mie Wong (Munro lab) presented an elegant system to test the function of a family of diverse Golgi-localized coiled-coil proteins. When different golgins were ectopically attached to mitochondria, specific classes of vesicles were redirected to mitochondria, clearly demonstrating a vesicle capture role for these proteins. A possible mechanism of lipid transfer from the ER to the PM was revealed by a structural study presented by Chris Schauder (Reinisch and de Camilli groups). The synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain of the ER–PM tether E-Syt2 forms a long hydrophobic channel that can harbor multiple phospholipid molecules. Two SMP domains form a stacked dimer to double the length of the channel. Continuing the theme of lipid composition in organelle identity, Cathy Jackson presented her group's results showing how amphipathic helices (AHs) of distinct chemistries specifically target different organelles through recognition of organelle lipid composition. Regulation of a lipid-binding AH of the Arf guanine nucleotide exchange factor (GEF) GBF1 mediates its localization to either the Golgi or lipid droplets. Looking at a related Arf GEF, yeast Sec7, Brian Richardson (Fromme lab) reported the first crystal structure of a noncatalytic region of these proteins. The N-terminal region is made up of α-helices forming a series of armadillo folds that form an extended platform. This structure provides insight into how this region is involved in dimerization and protein interactions. New vesicle tethering and targeting mechanisms were reported, with Chris Stroupe presenting how the homotypic fusion and protein sorting (HOPS)/class C Vps tethering complex connects specific membrane compartments. This complex binds to a Rab GTPase in one membrane and directly to a second, highly curved membrane through a curvature-sensing amphipathic lipid packing sensor (ALPS) motif. Margaret Heider (Munson lab) provided the most comprehensive view to date of the architecture of the exocyst complex, a multisubunit tether that targets secretory vesicles to the PM. The session ended with a talk by Christine Insinna-Kettenhofen (Westlake group) reporting on a new mechanism for targeting of vesicles to cilia mediated by the epidermal growth factor receptor substrate 15 homology domain–containing (EHD) proteins. These proteins are localized to vesicles, where they cooperate with the Rab8-Rab11 cascade and soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) to regulate morphological changes to the vesicle membrane important for fusion.

Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide–sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p’s functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion.

Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking.

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion.

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway.

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.

Variable cooperativity in SNARE-mediated membrane fusion.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.

Study of the Plant COPII Vesicle Coat Subunits by Functional Complementation of Yeast Saccharomyces cerevisiae Mutants

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23–Sec24 heterodimer following the subsequent recruitment of the Sec13–Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.