ABSTRACT
Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.
Subject(s)
Membrane Fusion , Saccharomyces cerevisiae Proteins , Vesicular Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , SNARE Proteins/metabolism , Qc-SNARE Proteins/metabolism , Vacuoles/metabolismABSTRACT
The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.
Subject(s)
Membrane Fusion , SNARE Proteins , Animals , SNARE Proteins/genetics , SNARE Proteins/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/metabolism , Mutation/genetics , Quality ControlABSTRACT
Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury.Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic-pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Sepsis , Animals , Mice , Adaptor Proteins, Signal Transducing/metabolism , Autophagy/physiology , Autophagy-Related Proteins/metabolism , Chemical and Drug Induced Liver Injury, Chronic/metabolism , Hepatocytes/metabolism , Lipopolysaccharides/pharmacology , Lysosomes/metabolism , Sepsis/complications , Sepsis/metabolism , SNARE Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/pharmacology , Ubiquitins/metabolismABSTRACT
Perfluorooctanoic acid (PFOA), a man-made chemical widely used in consumers, could cause male reproductive toxicity by disrupting blood-testis barrier (BTB) integrity. Autophagy in Sertoli cells is essential for regulation of spermatogenesis and BTB. However, it remains a mystery that whether PFOA-induced BTB injury is associated with autophagy in Sertoli cells. In this study, we found that PFOA dose-dependently disrupted tight junction (TJ) function in Sertoli cells in vivo and in vitro. Furthermore, the results from transmission electron microscopy, Western blot and immunofluorescence analysis revealed that PFOA induced the accumulation of autophagosome in testicular Sertoli cells as well as TM4 cells. Further study confirmed that autophagosome accumulation resulted from the blockage of autophagic degradation because of disruption of autophagosome and lysosome fusion via downregulation of the expression of α-SNAP. In parallel, the overexpressed MMP9 was also observed in vivo and in vitro. Conversely, overexpression of α-SNAP inhibited the expression of MMP9 in TM4 cells. In conclusion, PFOA blocks autophagic flux through downregulating the expression levels of α-SNAP in Sertoli cells, and then induces the accumulation of MMP9 leading to disruption of TJ function. This finding will provide clues for effective prevention and treatment of PFOA-induced male reproductive toxicity.
Subject(s)
Matrix Metalloproteinase 9 , Sertoli Cells , Humans , Male , Matrix Metalloproteinase 9/metabolism , Tight Junctions , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/pharmacology , Testis , Spermatogenesis , Autophagy , Blood-Testis BarrierABSTRACT
Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.
Subject(s)
Arabidopsis , Saccharomyces cerevisiae Proteins , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Autophagosomes/metabolism , Autophagy/physiology , Macroautophagy , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Endosomes/metabolism , SNARE Proteins/metabolism , Autophagy-Related Proteins/metabolism , Mammals/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.
Subject(s)
Membrane Fusion , SNARE Proteins , SNARE Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Protein Binding , Peptides/chemistryABSTRACT
Ca2+-triggered neurotransmitter release involves complex regulatory mechanisms, including a series of protein-protein interactions. Three proteins, synaptobrevin (VAMP), synaptosomal-associated protein of 25kDa (SNAP-25) and syntaxin, constitute the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex that plays key roles in controlling vesicle fusion and exocytosis. Many other proteins participate in the regulation of the processes via direct and/or indirect interaction with the SNARE complex. Although much effort has been made, the regulatory mechanism for exocytosis is still not completely clear. Accumulated evidence indicates that the small GTPase Rab3 and synaptotagmin proteins play important regulatory roles during vesicle fusion and neurotransmitter release. This review outlines our present understanding of the two regulatory proteins, with the focus on the interaction of Rab3 with synaptotagmin in the regulatory process.
Subject(s)
Monomeric GTP-Binding Proteins , Nerve Tissue Proteins , Synaptotagmins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Nerve Tissue Proteins/metabolism , Exocytosis/physiology , R-SNARE Proteins , Qa-SNARE Proteins/metabolism , Neurotransmitter Agents , Monomeric GTP-Binding Proteins/metabolism , Calcium/metabolismABSTRACT
Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in rat PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that the promotion of SNARE complex assembly by binding the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.
Subject(s)
Adaptor Proteins, Signal Transducing , Exocytosis , Membrane Fusion , Nerve Tissue Proteins , Synaptosomal-Associated Protein 25 , Vesicular Transport Proteins , Adaptor Proteins, Signal Transducing/metabolism , Animals , Exocytosis/physiology , Nerve Tissue Proteins/metabolism , Rats , SNARE Proteins/metabolism , Secretory Vesicles/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Vesicular Transport Proteins/metabolism , rab3 GTP-Binding Proteins/metabolism , Rabphilin-3AABSTRACT
Membrane fusion is driven by Sec17, Sec18, and SNARE zippering. Sec17 bound to SNAREs promotes fusion through its membrane-proximal N-terminal apolar loop domain. At its membrane-distal end, Sec17 serves as a high-affinity receptor for Sec18. At that distance from the fusion site, it has been unclear how Sec18 can aid Sec17 to promote fusion. We now report that Sec18, with ATPγS, lowers the Km of Sec17 for fusion. A C-terminal and membrane-distal Sec17 mutation, L291A,L292A, diminishes Sec17 affinity for Sec18. High levels of wild-type Sec17 or Sec17-L291AL292A show equivalent fusion without Sec18, but Sec18 causes far less fusion enhancement with low levels of Sec17-L291AL292A than with wild-type Sec17. Another mutant, Sec17-F21SM22S, has reduced N-loop apolarity. Only very high levels of this mutant protein support fusion, but Sec18 still lowers the apparent fusion Km for Sec17-F21SM22S. Thus Sec18 stimulates fusion through Sec17 and acts at the well-described interface between Sec18 and Sec17. ATP acts as a ligand to activate Sec18 for Sec17-dependent fusion, but ATP hydrolysis is not required. Even without SNAREs, Sec18 and Sec17 exhibit interdependent stable association with lipids, with several Sec17 bound for each Sec18 hexamer, explaining how Sec18 stabilization of surface-concentrated clusters of Sec17 lowers the Sec17 Km for assembly with SNAREs. Each of the associations, between SNARE complex, Sec18, Sec17, and lipid, helps assemble the fusion machinery.
Subject(s)
Membrane Fusion , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Ligands , Lipids , Membrane Fusion/physiology , Mutant Proteins/metabolism , SNARE Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.
Subject(s)
Calcium , Phosphatidylinositol 4,5-Diphosphate , Adaptor Proteins, Vesicular Transport/chemistry , Binding Sites , Calcium/metabolism , Exocytosis , Nerve Tissue Proteins/chemistry , Neurotransmitter Agents , SNARE Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Synaptotagmin I/chemistryABSTRACT
Synaptic exocytosis requires efficient SNARE complex assembly that is precisely regulated by multiple regulatory proteins. Increasing evidence suggests that Munc18-1 and Munc13-1 protect SNARE complex assembly in a manner resistant to NSF and α-SNAP. However, the protective mechanisms of Munc18-1 and Munc13-1 are not fully understood. Here, by analyzing two pathways of SNARE complex assembly (i.e., the Munc18 - Munc13-dependent pathway and the Munc18 - Munc13-independent pathway), we found that the Munc18 - Munc13-dependent pathway of SNARE complex assembly is resistant to NSF - α-SNAP. In this pathway, Munc18-1 and Munc13-1 each, independently, have protective effects. The protective effect of Munc18-1 relies on the interaction with the C-terminal part of Syb2 during the transition from the Munc18-1/Syx1 complex to the SNARE complex. Moreover, the protective effect of Munc13-1 is most likely attributed to its ability in templating the assembling SNAREs. In addition, we found that the Munc18 - Munc13-dependent pathway opposes the association of α-SNAP with the SNARE bundle, thus explaining how this pathway is resistant to NSF - α-SNAP disassembly. Although the above results were derived from the studies on SNARE complex in solution or in cis-configurations, instead of trans-configurations residing on the opposite membrane, our data could still help to understand the protective mechanism of Munc18-1 and Munc13-1 in SNARE-mediated synaptic exocytosis.
Subject(s)
Membrane Fusion , Munc18 Proteins , Carrier Proteins , Exocytosis , Munc18 Proteins/genetics , Munc18 Proteins/metabolism , SNARE Proteins/genetics , SNARE Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolismABSTRACT
Membrane fusion requires tethers, SNAREs of R, Qa, Qb, and Qc families, and chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. SNAREs have N-domains, SNARE domains that zipper into 4-helical RQaQbQc coiled coils, a short juxtamembrane (Jx) domain, and (often) a C-terminal transmembrane anchor. We reconstitute fusion with purified components from yeast vacuoles, where the HOPS protein combines tethering and SM functions. The vacuolar Rab, lipids, and R-SNARE activate HOPS to bind Q-SNAREs and catalyze trans-SNARE associations. With SNAREs initially disassembled, as they are on the organelle, we now report that R- and Qa-SNAREs require their physiological juxtamembrane (Jx) regions for fusion. Swap of the Jx domain between the R- and Qa-SNAREs blocks fusion after SNARE association in trans. This block is bypassed by either Sec17, which drives fusion without requiring complete SNARE zippering, or transmembrane-anchored Qb-SNARE in complex with Qa. The abundance of the trans-SNARE complex is not the sole fusion determinant, as it is unaltered by Sec17, Jx swap, or the Qb-transmembrane anchor. The sensitivity of fusion to Jx swap in the absence of a Qb transmembrane anchor is inherent to the SNAREs, because it remains when a synthetic tether replaces HOPS.
Subject(s)
SNARE Proteins , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/metabolism , Membrane Fusion/physiology , Proteolipids/metabolism , Qa-SNARE Proteins/metabolism , SNARE Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vacuoles/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Loss of the furin cleavage motif in the SARS-CoV-2 spike protein reduces the virulence and transmission of SARS-CoV-2, suggesting that furin is an attractive antiviral drug target. However, lack of understanding of the regulation of furin activity has largely limited the development of furin-based therapeutic strategies. Here, we find that alpha-soluble NSF attachment protein (α-SNAP), an indispensable component of vesicle trafficking machinery, inhibits the cleavage of SARS-CoV-2 spike protein and other furin-dependent virus glycoproteins. SARS-CoV-2 infection increases the expression of α-SNAP, and overexpression of α-SNAP reduces SARS-CoV-2 infection in cells. We further reveal that α-SNAP is an interferon-upregulated furin inhibitor that inhibits furin function by interacting with its P domain. Our study demonstrates that α-SNAP, in addition to its role in vesicle trafficking, plays an important role in the host defense against furin-dependent virus infection and therefore could be a target for the development of therapeutic options for COVID-19. IMPORTANCE Some key mutations of SARS-CoV-2 spike protein, such as D614G and P681R mutations, increase the transmission or pathogenicity by enhancing the cleavage efficacy of spike protein by furin. Loss of the furin cleavage motif of SARS-CoV-2 spike protein reduces the virulence and transmission, suggesting that furin is an attractive antiviral drug target. However, lack of understanding of the regulation of furin activity has largely limited the development of furin-based therapeutic strategies. Here, we found that in addition to its canonical role in vesicle trafficking, alpha-soluble NSF attachment protein (α-SNAP) plays an important role in the host defense against furin-dependent virus infection. we identified that α-SNAP is a novel interferon-upregulated furin inhibitor and inhibits the cleavage of SARS-CoV-2 spike protein and other furin-dependent virus glycoproteins by interacting with P domain of furin. Our study demonstrates that α-SNAP could be a target for the development of therapeutic options for COVID-19.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/metabolism , SARS-CoV-2/metabolism , Furin/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Interferons/metabolism , Carrier Proteins , Antiviral Agents , Glycoproteins/metabolismABSTRACT
This study pursued the hypothesis that wild plant germplasm accessions carrying alleles of interest can be identified using available single nucleotide polymorphism (SNP) genotypes for particular alleles of other (unlinked) genes that contribute to the trait of interest. The soybean cyst nematode (SCN, Heterodera glycines [HG]) resistance locus Rhg1 is widely used in farmed soybean [Glycine max (L.) Merr.]. The two known resistance-conferring haplotypes, rhg1-a and rhg1-b, typically contain three or seven to 10 tandemly duplicated Rhg1 segments, respectively. Each Rhg1 repeat carries four genes, including Glyma.18G022500, which encodes unusual isoforms of the vesicle-trafficking chaperone α-SNAP. Using SoySNP50K data for NSFRAN07 allele presence, we discovered a new Rhg1 haplotype, rhg1-ds, in six accessions of wild soybean, Glycine soja Siebold & Zucc. (0.5% of the â¼1,100 G. soja accessions in the USDA collection). The α-SNAP encoded by rhg1-ds is unique at an important site of amino acid variation and shares with the rhg1-a and rhg1-b α-SNAP proteins the traits of cytotoxicity and altered N-ethylmaleimide sensitive factor (NSF) protein interaction. Copy number assays indicate three repeats of rhg1-ds. G. soja PI 507613 and PI 507623 exhibit resistance to HG type 2.5.7 SCN populations, in part because of contributions from other loci. In a segregating F2 population, rhg1-b and rhg1-ds made statistically indistinguishable contributions to resistance to a partially virulent HG type 2.5.7 SCN population. Hence, the unusual multigene copy number variation Rhg1 haplotype was present but rare in ancestral G. soja and was present in accessions that offer multiple traits for SCN resistance breeding. The accessions were initially identified for study based on an unlinked SNP.
Subject(s)
Disease Resistance , Tylenchoidea , Animals , DNA Copy Number Variations , Disease Resistance/genetics , Glycine , Haplotypes , Plant Breeding , Plant Diseases/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Glycine max/genetics , Tylenchoidea/metabolismABSTRACT
We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.
Subject(s)
Microtubule-Associated Proteins , Receptors, Opioid, kappa , Animals , Cricetinae , Cricetulus , N-Ethylmaleimide-Sensitive Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolismABSTRACT
Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.
Subject(s)
Adenosine Triphosphatases/genetics , Membrane Fusion , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/physiology , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Vesicular Transport Proteins/genetics , Adenosine Triphosphatases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
Molecular steps that activate store-operated calcium entry (SOCE) via Orai channel supramolecular complex remain incompletely defined. We have earlier shown that α-SNAP regulates the on-site functional assembly and calcium selectivity of Orai1 channels. Here we investigate the molecular basis of its association with Orai, Stim and find that the affinity of α-SNAP for Orai and Stim is substantially higher than previously reported affinities between Stim and Orai sub-domains. α-SNAP binds the coiled-coil 3 (CC3) sub-domain of Stim1. Mutations of Tryptophan 430 in Stim1-CC3 disrupted α-SNAP association and SOCE, demonstrating a novel α-SNAP dependent function for this crucial subdomain. Further, α-SNAP binds the hinge region near the C-terminus of Orai1 and an additional broad region near the N-terminus and Valine 262 and Leucine 74 were necessary for these respective interactions, but not Orai, Stim co-clustering. Thus, high affinity interactions with α-SNAP are necessary for imparting functionality to Stim, Orai clusters and induction of SOCE.
Subject(s)
Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , ORAI1 Protein/genetics , ORAI1 Protein/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolism , Animals , Binding Sites , Cell Line , Cloning, Molecular , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Knockout Techniques , HEK293 Cells , Humans , Mice , Mutation , Neoplasm Proteins/chemistry , ORAI1 Protein/chemistry , Protein Binding , Stromal Interaction Molecule 1/chemistryABSTRACT
Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.
Subject(s)
Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositols/metabolism , SNARE Proteins/metabolism , Catalysis , Inositol Phosphates/metabolism , Intracellular Membranes/metabolism , Membrane Fusion/physiology , Molecular Chaperones/metabolism , Phosphates/metabolism , Phosphatidic Acids/metabolism , Protein Binding , Proteolipids/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Vacuoles/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.
Subject(s)
Frontotemporal Dementia/metabolism , Models, Molecular , Muscular Dystrophies, Limb-Girdle/metabolism , Mutation , Myositis, Inclusion Body/metabolism , Osteitis Deformans/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Valosin Containing Protein/genetics , Frontotemporal Dementia/genetics , Humans , Microscopy, Electron, Transmission , Muscular Dystrophies, Limb-Girdle/genetics , Myositis, Inclusion Body/genetics , Osteitis Deformans/genetics , Protein Conformation , Valosin Containing Protein/metabolismABSTRACT
Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.