Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation.

Microtubule-associated protein tau is a central factor in Alzheimer's disease and other tauopathies. However, the physiological functions of tau are unclear. Here, we used proximity-labelling proteomics to chart tau interactomes in primary neurons and mouse brains in vivo. Tau interactors map onto pathways of cytoskeletal, synaptic vesicle and postsynaptic receptor regulation and show significant enrichment for Parkinson's, Alzheimer's and prion disease. We find that tau interacts with and dose-dependently reduces the activity of N-ethylmaleimide sensitive fusion protein (NSF), a vesicular ATPase essential for AMPA-type glutamate receptor (AMPAR) trafficking. Tau-deficient (tau-/- ) neurons showed mislocalised expression of NSF and enhanced synaptic AMPAR surface levels, reversible through the expression of human tau or inhibition of NSF. Consequently, enhanced AMPAR-mediated associative and object recognition memory in tau-/- mice is suppressed by both hippocampal tau and infusion with an NSF-inhibiting peptide. Pathologic mutant tau from mouse models or Alzheimer's disease significantly enhances NSF inhibition. Our results map neuronal tau interactomes and delineate a functional link of tau with NSF in plasticity-associated AMPAR-trafficking and memory.

Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF.

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from NSF/α-SNAP-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy.

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines.

ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.

The canonical α-SNAP is essential for gametophytic development in Arabidopsis.

The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA+ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.

Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites.

An atypical Phytophthora sojae RxLR effector manipulates host vesicle trafficking to promote infection.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF.

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Ca2+-dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

Metabotropic Glutamate Receptors Modulate Exocytotic Tau Release and Propagation.

Using synaptosomes purified from the brains of two transgenic mouse models overexpressing mutated human tau (TgP301S and Tg4510) and brains of patients with sporadic Alzheimer's disease, we showed that aggregated and hyperphosphorylated tau was both present in purified synaptosomes and released in a calcium- and synaptosome-associated protein of 25 kDa (SNAP25)-dependent manner. In all mouse and human synaptosomal preparations, tau release was inhibited by the selective metabotropic glutamate receptor 2/3 (mGluR2/3) agonist LY379268, an effect prevented by the selective mGlu2/3 antagonist LY341495. LY379268 was also able to block pathologic tau propagation between primary neurons in an in vitro microfluidic cellular model. These novel results are transformational for our understanding of the molecular mechanisms mediating tau release and propagation at synaptic terminals in Alzheimer's disease and suggest that these processes could be inhibited therapeutically by the selective activation of presynaptic G protein-coupled receptors. SIGNIFICANCE STATEMENT: Pathological tau release and propagation are key neuropathological events underlying cognitive decline in Alzheimer's disease patients. This paper describes the role of regulated exocytosis, and the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein SNAP25, in mediating tau release from rodent and human synaptosomes. This paper also shows that a selective mGluR2/3 agonist is highly effective in blocking tau release from synaptosomes and tau propagation between neurons, opening the way to the discovery of novel therapeutic approaches to this devastating disease.

Vesicle trafficking with snares: a perspective for autism.

Vesicle-mediated membrane traffic is the mechanism fundamental to many biological events, especially the release of neurotransmitters. The main proteins of the mechanism that mediates membrane fusion in vesicle-mediated membrane traffic are N-ethylmaleimide sensitive factor (NSF) supplemental protein (SNAP) receptor (SNAREs) proteins. SNAREs are classified into vesicle-associated SNAREs (vesicle-SNAREs/v-SNAREs) and target membrane-associated SNAREs (target-SNARE/t-SNAREs). Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by many symptoms, especially complications in social communication and stereotypical behaviours. Defects in synaptogenesis and neurotransmission, oxidative stress, and developmental defects in the early stages of development are defined in the pathogenesis of the disease. SNARE proteins are on the basis of synaptogenesis and neurotransmission. Although the formation mechanisms and underlying causes of the SNARE complex are not fully understood, expression differences, polymorphisms, abnormal expressions or dysfunctions of the proteins that make up the SNARE complex have been associated with many neurodevelopmental diseases, including autism. Further understanding of SNARE mechanisms is crucial both for understanding ASD and for developing new treatments. In this review, the formation mechanisms of the SNARE complex and the roles of various factors involved in this process are explained. In addition, a brief evaluation of clinical and basic studies on the SNARE complex in autism spectrum disorders was made.

LRRK2 G2019S kinase activity triggers neurotoxic NSF aggregation.

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. The LRRK2 G2019S mutation is one of the major determinants of familial Parkinson's disease cases and leads to late-onset Parkinson's disease with pleomorphic pathology, including α-synuclein accumulation and deposition of protein inclusions. We demonstrated that LRRK2 phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from patients with Parkinson's disease carrying the G2019S variant, and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Of note, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S bacterial artificial chromosome (BAC) mice. We suggest that LRRK2 G2019S pathological phosphorylation impacts on NSF biochemical properties, thus causing the formation of cytotoxic protein inclusions.

Does GEC1 Enhance Expression and Forward Trafficking of the Kappa Opioid Receptor (KOR) via Its Ability to Interact with NSF Directly?

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.

Mechanistic insights into the recycling machine of the SNARE complex.

Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

Syntaxin 13, a genetic modifier of mutant CHMP2B in frontotemporal dementia, is required for autophagosome maturation.

Phagophore maturation is a key step in the macroautophagy pathway, which is critical in many important physiological and pathological processes. Here we identified Drosophila N-ethylmaleimide-sensitive fusion protein 2 (dNSF2) and soluble NSF attachment protein (Snap) as strong genetic modifiers of mutant CHMP2B, an ESCRT-III component that causes frontotemporal dementia and autophagosome accumulation. Among several SNAP receptor (SNARE) genes, Drosophila syntaxin 13 (syx13) exhibited a strong genetic interaction with mutant CHMP2B. Knockdown of syntaxin 13 (STX13) or its binding partner Vti1a in mammalian cells caused LC3-positive puncta to accumulate and blocks autophagic flux. STX13 was present on LC3-positive phagophores induced by rapamycin and was highly enriched on multilamellar structures induced by dysfunctional ESCRT-III. Loss of STX13 also caused the accumulation of Atg5-positive puncta and the formation of multilamellar structures. These results suggest that STX13 is a genetic modifier of ESCRT-III dysfunction and participates in the maturation of phagophores into closed autophagosomes.

An atypical N-ethylmaleimide sensitive factor enables the viability of nematode-resistant Rhg1 soybeans.

N-ethylmaleimide sensitive factor (NSF) and α-soluble NSF attachment protein (α-SNAP) are essential eukaryotic housekeeping proteins that cooperatively function to sustain vesicular trafficking. The "resistance to Heterodera glycines 1" (Rhg1) locus of soybean (Glycine max) confers resistance to soybean cyst nematode, a highly damaging soybean pest. Rhg1 loci encode repeat copies of atypical α-SNAP proteins that are defective in promoting NSF function and are cytotoxic in certain contexts. Here, we discovered an unusual NSF allele (Rhg1-associated NSF on chromosome 07; NSFRAN07 ) in Rhg1+ germplasm. NSFRAN07 protein modeling to mammalian NSF/α-SNAP complex structures indicated that at least three of the five NSFRAN07 polymorphisms reside adjacent to the α-SNAP binding interface. NSFRAN07 exhibited stronger in vitro binding with Rhg1 resistance-type α-SNAPs. NSFRAN07 coexpression in planta was more protective against Rhg1 α-SNAP cytotoxicity, relative to WT NSFCh07 Investigation of a previously reported segregation distortion between chromosome 18 Rhg1 and a chromosome 07 interval now known to contain the Glyma.07G195900 NSF gene revealed 100% coinheritance of the NSFRAN07 allele with disease resistance Rhg1 alleles, across 855 soybean accessions and in all examined Rhg1+ progeny from biparental crosses. Additionally, we show that some Rhg1-mediated resistance is associated with depletion of WT α-SNAP abundance via selective loss of WT α-SNAP loci. Hence atypical coevolution of the soybean SNARE-recycling machinery has balanced the acquisition of an otherwise disruptive housekeeping protein, enabling a valuable disease resistance trait. Our findings further indicate that successful engineering of Rhg1-related resistance in plants will require a compatible NSF partner for the resistance-conferring α-SNAP.

New gadget in the membrane trafficking toolbox: A novel inhibitor of SNARE priming.

NSF (N-ethylmaleimide sensitive factor) and its yeast counterpart Sec18 are highly conserved homohexameric proteins that play vital roles in eukaryotic membrane trafficking. Sec18 functions by disrupting SNARE complexes formed in cis, on the same membrane. However, the molecular mechanisms of this process are poorly understood, in large part due to the lack of selective, reversible inhibitors. A new study by Sparks et al. now reports a small molecule that appears to selectively inhibit Sec18 action in an in vitro assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming.

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.

Functional characterization of UBXN-6, a C-terminal cofactor of CDC-48, in C. elegans.

CDC-48 is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities. Its functional diversity is determined by differential binding of a variety of cofactors. In this study, we analyzed the physiological role of a CDC-48 cofactor UBXN-6 in Caenorhabditis elegans. The amount of UBXN-6 was markedly increased upon starvation, but not with the treatment of tunicamycin and rapamycin. The induction upon starvation is a unique characteristic for UBXN-6 among C-terminal cofactors of CDC-48. During starvation, lysosomal activity is triggered for rapid clearance of cellular materials. We observed the lysosomal activity by monitoring GLO-1::GFP, a marker for lysosome-related organelles. We found that more puncta of GLO-1::GFP were observed in the ubxn-6 deletion mutant after 12 h starvation compared with the wild-type strain. Taken together, we propose that UBXN-6 is involved in clearance of cellular materials upon starvation in C. elegans.

A connection between reversible tyrosine phosphorylation and SNARE complex disassembly activity of N-ethylmaleimide-sensitive factor unveiled by the phosphomimetic mutant N-ethylmaleimide-sensitive factor-Y83E.

N-ethylmaleimide-sensitive factor (NSF) disassembles fusion-incompetent cis soluble-NSF attachment protein receptor (SNARE) complexes making monomeric SNAREs available for subsequent trans pairing and fusion. In most cells the activity of NSF is constitutive, but in Jurkat cells and sperm it is repressed by tyrosine phosphorylation; the phosphomimetic mutant NSF-Y83E inhibits secretion in the former. The questions addressed here are if and how the NSF mutant influences the configuration of the SNARE complex. Our model is human sperm, where the initiation of exocytosis (acrosome reaction (AR)) de-represses the activity of NSF through protein tyrosine phosphatase 1B (PTP1B)-mediated dephosphorylation. We developed a fluorescence microscopy-based method to show that capacitation increased, and challenging with an AR inducer decreased, the number of cells with tyrosine-phosphorylated PTP1B substrates in the acrosomal domain. Results from bioinformatic and biochemical approaches using purified recombinant proteins revealed that NSF-Y83E bound PTP1B and thereupon inhibited its catalytic activity. Mutant NSF introduced into streptolysin O-permeabilized sperm impaired cis SNARE complex disassembly, blocking the AR; subsequent addition of PTP1B rescued exocytosis. We propose that NSF-Y83E prevents endogenous PTP1B from dephosphorylating sperm NSF, thus maintaining NSF's activity in a repressed mode and the SNARE complex unable to dissociate. The contribution of this paper to the sperm biology field is the detection of PTP1B substrates, one of them likely being NSF, whose tyrosine phosphorylation status varies during capacitation and the AR. The contribution of this paper to the membrane traffic field is to have generated direct evidence that explains the dominant-negative role of the phosphomimetic mutant NSF-Y83E.