Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Prinslow, Eric A; Stepien, Karolina P.; Pan, Yun Zu; Xu, Junjie; Rizo, Josep

doi:10.7554/elife.38880

Cited by 63 publications

(109 citation statements)

References 81 publications

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“…Sly1 reduces the rate of SNARE complex disassembly by Sec17 and Sec18 (Lobingier et al, 2014). This is consistent with studies of Vps33 and Munc18-1, showing that these SMs protect assembled trans-SNARE complexes from premature disassembly by Sec17 and Sec18 (Lobingier et al, 2014;Prinslow et al, 2019;Schwartz et al, 2017;Song et al, 2017;Stepien et al, 2019;Xu et al, 2010;Duan et 375 al., submitted). Thus we reiterate our previous suggestions (Lobingier et al, 2014;Schwartz et al, 2017) that SM proteins are, sensu stricto, enzymes.…”

supporting

confidence: 72%

“…Also as expected for true enzymes, SMs prevent off-pathway reactions (e.g., assembly of non-cognate SNARE complexes, or of cisrather than trans-complexes). They achieve this increase in specificity through kinetic partitioning within the forward assembly pathway (Hardy and Randall, 1991;Lai et al, 2017;Lambright et al, 1994;Peng and Gallwitz, 2002), and through kinetic proofreading of incorrect 385 SNARE assemblies, since SMs selectively protect cognate SNARE complexes from premature disassembly by proofreading enzymes, while non-cognate complexes are efficiently disassembled (Choi et al, 2018;Lobingier et al, 2014;Prinslow et al, 2019;Schwartz et al, 2017;Song et al, 2017;Xu et al, 2010).…”

mentioning

confidence: 99%

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Golgi SM protein Sly1 promotes productivetrans-SNARE complex assembly through multiple mechanisms

Duan

Gao

Banfield

2020

Preprint

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SNARE chaperones of the Sec1/mammalian Unc-18 (SM) family have critical roles in SNAREmediated membrane fusion. Using SNARE and Sly1 mutants, and a new in vitro assay of fusion, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles 5 to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) preferential nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and we present evidence that close-range tethering is particularly important for trans-complex assembly when cis-SNARE assembly is a competing process. In addition, the autoinhibitory N-terminal Habc domain of Sed5 has at least two 10 positive activities: the Habc domain is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. Remarkably, "split Sed5," with the Habc domain present only as a soluble fragment, is functional both in vitro and in vivo.In addition to SNAREs and tethering factors, proteins of the Sec1/mammalian Unc-18 (SM) family have critical roles in SNARE-mediated fusion (Carr and Rizo, 2010;Rizo and Sudhof, 2012;Sudhof and Rothman, 2009). The first SM proteins identified through genetic 35 screens were Vps33a (carnation in Drosophila) and Saccharomyces cerevisiae Sec1 (UNC-18 in Caenorabditis elegans; Munc18-1 or nSec1 in mammals; Novick et al., 1979;Patterson, 1932).Despite their early identification and clear importance, and despite major efforts by many laboratories, the general mechanisms of SM function are only now emerging. All SM proteins exhibit strong evolutionary and structural homology, but they interact with cognate SNARE 40 proteins in very different ways. For example, yeast Sly1, yeast Vps45, and Munc18-1 all interact with short N-peptides at the amino termini of their cognate Qa-SNARE proteins (Bracher and Weissenhorn, 2002;Carpp et al., 2006;Dulubova et al., 2002;Furgason et al., 2009; Grabowski and Gallwitz, 1997;Yamaguchi et al., 2002). In contrast, Qa-SNARE N-peptide interactions do not occur with human or yeast Vps33, or with yeast Sec1 (Baker et al., 2015; Dulubova et al., 45 2001;Lobingier and Merz, 2012;Togneri et al., 2006). We have called SM proteins that interact with Qa-SNARE N-peptides Class I, and those that do not, Class II (Lobingier and Merz, 2012).Early structural and biochemical studies revealed that Munc18-1 tightly binds the Qa-SNARE Syntaxin-1A in its closed conformation, suggesting an inhibitory role for Munc18-1. (Dulubova et al., 1999;Misura et al., 2000;Yang et al., 2000). However, the emerging consensus 50 is that the core and evolutionarily conserved role of SM proteins is positive, rather than inhibitory. Specifically, SM proteins are hypothesized to nucleate and stabilize fusioncompetent trans-SNARE complexes (Carr and Rizo, 2010;Sudhof and Rothman, 2009;Toonen and Verhage, 2003;Yoon and Munson, 2018). A breakthrough was achieved in 2015 with two structure...

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supporting

confidence: 72%

mentioning

confidence: 99%

Golgi SM protein Sly1 promotes productivetrans-SNARE complex assembly through multiple mechanisms

Duan

Gao

Banfield

2020

Preprint

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“…Central for membrane fusion are the neuronal SNAP receptors (SNAREs) syntaxin‐1, SNAP‐25, and synaptobrevin, which form a tight SNARE complex that consists of a four‐helix bundle and brings the two membranes together . N‐ethylmaleimide sensitive factor (NSF) and soluble NSF adaptor proteins (SNAPs) disassemble SNARE complexes to recycle the SNAREs, whereas Munc18‐1 and Munc13s coordinate SNARE complex assembly in an NSF‐SNAP‐resistant manner, and synaptotagmin‐1 acts as the Ca 2+ sensor that triggers fast, synchronous release through interactions with phospholipids and the SNAREs, in a tight interplay with complexins …”

Section: Introductionmentioning

confidence: 99%

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

et al. 2020

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Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with syntaxin-1 folded into a selfinhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin-1 via interactions with the linker. K E Y W O R D S conformational transition, membrane fusion, Munc13, Munc18, neurotransmitter release, SNAREs, syntaxin-1

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“…8). These findings can be readily explained by results from reconstitution assays showing that Munc18-1 and Munc13-1 are normally essential to form trans-SNARE complexes in the presence of NSF and αSNAP, because Munc18-1 and Munc13-1 orchestrate SNARE complex assembly in an NSF-αSNAP-resistant manner 53, 54, 61 . The requirement of Munc13-1 for liposome fusion can be partially bypassed by the open syntaxin mutation (Fig.…”

Section: Discussionmentioning

confidence: 99%

Open syntaxin overcomes synaptic transmission defects in diverse C. elegans exocytosis mutants

Tien

Huang

et al. 2020

Preprint

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Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a 'closed' conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibited enhanced spontaneous and evoked exocytosis compared to wild-type animals. Unexpectedly, the open syntaxin KI partially suppressed exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca 2+ channel alpha-subunit, and unc-31/CAPS in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravated the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states. Keywords: exocytosis, vesicle priming, SNARE, synaptotagmin, Munc18/UNC-18, Munc13/UNC-13 forming a tight SNARE complex 1 that consists of a four-helix bundle 2,3 . This four-helix bundle is believed to be partially formed before Ca 2+ influx [reviewed in 4 ]. Ca 2+ , acting through synaptotagmin-1 5,6 , triggers full zippering of the SNARE complex, and ultimately results in fusion of the vesicle with the plasma membrane. A biochemical step preceding the full fusion is often referred to as "priming". It involves formation of the fusion-competent state of synaptic vesicles just prior to Ca 2+ entry. The priming process is tightly controlled by multiple proteins, including Munc18-1/UNC-18 7-9 , Munc13-1/2/UNC-13 10-12 , Tomosyn 13-15 and RIM/UNC-10 16,17 . However, the precise mechanisms through which these proteins mediate priming is still unclear. Syntaxin/UNC-64 is a key SNARE protein that is crucial for exocytosis. It exists in an open and a closed conformation, the latter forming a tight complex with Munc18-1/UNC-18 18 . The binding of closed syntaxin and Munc18-1/UNC-18 has been shown to be critical for the level and plasmalemmal localization of syntaxin 7,19-22 . However, when held closed by Munc18-1/UNC-18, syntaxin is unable to interact with synaptobrevin and SNAP-25 to form SNARE complexes 23,24 . Munc13-1/UNC-13 was postulated to open syntaxin and consequently promote SNARE complex assembly and subsequent exocytosis at axon terminals. This important hypothesis originated from studies in which multi-copy over-expression of a constitutively open form of syntaxin/UNC-64 in C. elegans partially restored motility and strongly rescued synaptic vesicle exocytosis defects of unc-13 null worms 25 . The rescue was suggested to be due to a specific genetic interaction between unc-13 and syntaxin, as expression of the open syntaxin in unc-64 null (js21) mutant 26 did not enhance exocytosis compar...

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Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

Cited by 63 publications

References 81 publications

Golgi SM protein Sly1 promotes productivetrans-SNARE complex assembly through multiple mechanisms

Golgi SM protein Sly1 promotes productivetrans-SNARE complex assembly through multiple mechanisms

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

Open syntaxin overcomes synaptic transmission defects in diverse C. elegans exocytosis mutants

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