Tethering guides fusion-competent
            <i>trans</i>
            -SNARE assembly

Song, Hongki; Wickner, William

doi:10.1073/pnas.1907640116

Cited by 24 publications

(59 citation statements)

References 49 publications

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“…This model predicts that Munc13‐1 cannot be replaced by a factor that merely bridges membranes and cannot interact specifically with syntaxin‐1. To test this prediction, we took advantage of the recent observation that a dimeric fusion protein of GST with a PX domain that binds tightly to PI3P catalyzes fusion between liposomes bearing the yeast vacuolar homolog of synaptobrevin and liposomes bearing the cognate syntaxin‐1‐SNAP‐25 homologs when both liposome populations contained PI3P . Thus, we prepared syntaxin‐1‐SNAP‐25 liposomes and synaptobrevin liposomes containing 1% PI3P (referred to as T*‐liposomes and V*‐liposomes, respectively), and investigated whether Munc13‐1 C 1 C 2 BMUNC 2 C can be functionally replaced by the GST‐PX fusion protein.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

“…All mutant proteins were purified as the wild type proteins. Purified fusion protein of GST with the PX domain of Vam7 was a kind gift from William Wickner. To obtain uniformly 15 N‐labeled proteins, we used M9 minimal expression media with 15 NH 4 Cl as the sole nitrogen source (1 g/L).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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“…Tethering is a prerequisite for SNAREs to assemble in trans in a productive conformation (35). Tethering is normally performed by a Rab and its effector (33).…”

Section: Resultsmentioning

confidence: 99%

Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

Jun

Wickner

2019

Proc. Natl. Acad. Sci. U.S.A.

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Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

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“…To study the functional 140intermediates in SNARE complex assembly, we assayed fusion without an added 141 tether, with tethering by the physiological and multifunctional HOPS complex bound to 142 the Rab Ypt7 on each membrane, or with a simple synthetic tether. Our synthetic tether 143 consists of dimeric glutathione S-transferase (GST) fused to a PX domain that can bind 144 to PtdIns3P in each proteoliposomal membrane (Song and Wickner, 2019). 145Proteoliposomes bearing Ypt7 and R-SNARE with lumenally entrapped biotinylated 146 phycoerythrin were mixed with proteoliposomes bearing Ypt7 and the 3 Q-SNAREs with 147 entrapped Cy5-streptavidin.…”

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confidence: 99%

“…This was not seen 186 with GST-PX and 2Q-SNARE proteoliposomes lacking Qa or Qb and supplemented 187with sQa or sQb, respectively (Figure 1C, D), suggesting that these assembly events 188 are either kinetically too slow or thermodynamically unfavorable. 189As a second, complementary assay for spontaneous assembly of functional 3Q-SNARE 191 complex, we employed the R-SNARE without its membrane anchor, termed soluble-R 192 (sR), a known fusion inhibitor(Thorngren et al, 2004;Zick and Wickner, 2014; Song 193 andWickner, 2019). Inhibition by sR can employ two mechanisms: 1. sR may compete 194 for the conserved R-SNARE binding groove on the Vps33 subunit of HOPS (Baker et 195 al., 2015).…”

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confidence: 99%

HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE

Song

Orr

Harner

et al. 2019

Preprint

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26Vacuole fusion requires SNAREs, Sec17/18, a Rab, and HOPS. We find that co-27 incubation of HOPS, proteoliposomes bearing the Rab and R-SNARE, and 28 proteoliposomes with the Rab and any two Q-SNAREs yields a trans complex which 29 includes these 3 SNAREs. The missing Q-SNARE then triggers a burst of fusion, 30 indicating that each HOPS, R-, and QxQy-SNARE trans-complex is an activated 31 intermediate for functional Qz-SNARE incorporation. HOPS can assemble activated 32 fusion intermediates because it recognizes each of the four SNAREs, binding them 33 independently. HOPS-dependent fusion is saturable for each Q-SNARE, indicating 34 saturable functional sites on HOPS. Though a nonspecific tether allows fusion with pre-35 assembled Q-SNAREs, only HOPS catalyzes fusion when the Q-SNAREs are not pre-36 assembled by ushering each Q-SNARE into a functional complex. In contrast, there is 37 little spontaneous functional assembly of the 3 Q-SNAREs. HOPS thus recognizes each 38 of the 4 SNAREs to assemble a versatile set of activated fusion intermediates. 39 40 41 42 Hughson, 2016). SNARE (soluble N-ethylmaleimide-sensitive-factor attachment 48 receptor) proteins are found on both fusion partners, either in cis-SNARE complexes if 49 all are anchored to one membrane or trans-SNARE complexes if anchored to two 50 apposed membranes. SNAREs have heptad-repeat SNARE domains of approximately 51 50 aminoacyl residues with a central arginyl (R) or glutaminyl (Q) residue. SNAREs are 52 grouped by sequence homology into four families, R, Qa, Qb, and Qc (Fasshauer et al., 53 1998). SNARE complexes have one member each of the R, Qa, Qb, and Qc families, 54 with their -helical SNARE domains wrapped together in a coiled coil (Sutton et al., 55 1998). This 4-SNARE bundle is stabilized by the interior disposition of apolar residues, 56 with the exception of 1 arginyl and 3 glutaminyl residues in the center of the SNARE 57 domain, termed the 0-layer. SNARE complex assembly can be promoted by 58 Sec1/Munc18 (SM) family proteins (Baker et al., 2015; Jiao et al., 2018; Rizo and 59 Südhof, 2012). Disassembly of the post-fusion cis-SNARE complexes is catalyzed by 60 the ATP-driven chaperone Sec18/NSF, stimulated by its co-chaperone Sec17/SNAP 61 (White et al., 2018). Sec17 and Sec18 also function earlier, stimulating the fusion of 62 docked membranes (Song et al., 2017; Zick et al., 2015). Fusion also requires fatty acyl 63 fluidity (Zick and Wickner, 2016), acidic lipids and phosphoinositides to promote the 64 binding of peripheral membrane fusion proteins (Cheever et al., 2001; Mima and 65 Wickner, 2009; Orr et al., 2015), and nonbilayer-prone lipids (Zick et al., 2014) to enable 66 the bilayer rearrangements which are essential for fusion. 67 68 We study membrane fusion mechanisms with the vacuole (lysosome) of S. cerevisiae. 69 Vacuoles undergo constant fission and fusion, regulated by growth medium osmolarity. 70 Mutations which block fusion allow continued fission, resulting in a visibly altered 71 vacuole morphology which allowed s...

show abstract

Tethering guides fusion-competent trans -SNARE assembly

Cited by 24 publications

References 49 publications

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

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

Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE

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