Soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptors (SNAREs) are hypothesized to trigger membrane fusion by complexing in trans through their membrane-distal N termini and zippering toward their membrane-embedded C termini, which in turn drives the two membranes together. In this study, we use a set of truncated SNAREs to trap kinetically stable, partially zipped trans-SNARE complexes on intact organelles in the absence of hemifusion and content mixing. We show that the C-terminal zippering of SNARE cytoplasmic domains controls the onset of lipid mixing but not the subsequent transition from hemifusion to full fusion. Moreover, we find that a partially zipped nonfusogenic trans-complex is rescued by Sec17, a universal SNARE cochaperone. Rescue occurs independently of the Sec17-binding partner Sec18, and it exhibits steep cooperativity, indicating that Sec17 engages multiple stalled trans-complexes to drive fusion. These experiments delineate distinct functions within the trans-complex, provide a straightforward method to trap and study prefusion complexes on native membranes, and reveal that Sec17 can rescue a stalled, partially zipped trans-complex.

In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cellspecific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3-to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications.KEYWORDS CRISPR/Cas9; high-throughput; gene tagging; plasmid toolkit T HE clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized genome editing in nearly all model systems, including Caenorhabditis elegans (Frøkjaer-Jensen 2013;Doudna and Charpentier 2014;Xu 2015). The Cas9 protein cuts genomic DNA at sites that match an 20-nucleotide guide RNA sequence (Gasiunas et al. 2012;Jinek et al. 2012). Error-prone repair of the break can create point mutations, small indels, or large deletions at the cut site (Cho et al. 2013;Cong et al. 2013;Friedland et al. 2013;Jinek et al. 2013;van Schendel et al. 2015). However, the true power of CRISPR/Cas9 for genome editing lies in the insertion of exogenous DNA sequences, such as genetically encoded protein tags (Katic and Grosshans 2013;Lo et al. 2013;Mali et al. 2013;Tzur et al. 2013). To insert an exogenous sequence, one must simply supply a repair template with homology arms that flank the cut site. The cell uses homologybased repair to heal the break and copy the exogenous DNA into the cut site.Although in theory CRISPR/Cas9 makes it easy to insert exogenous sequences into a genome, practical limitations have prevented high-throughput implementation. Two critical limiting factors are (1) the time and expense required to build both the repair template a...

Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP-or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17 L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17 F21S,M22S , with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17 F21S,M22S , interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.I ntracellular vesicular traffic between organelles is the basis of cell growth, hormone secretion, and neurotransmission. At each step of exocytic and endocytic trafficking, membranes dock and fuse, mixing their lipids and luminal contents while keeping them separate from the cytosol. Families of proteins, conserved from yeast to humans, mediate docking and fusion. Fusion requires Rab family GTPases and "effector" proteins that bind to a Rab in its active, GTP-bound state (1). Among the effectors are large, organelle-specific tethering complexes. Fusion requires SNARE proteins and their chaperones. SNAREs (2) are proteins that can "snare" (bind to) each other, in cis (when anchored to the same membrane) or in trans (when anchored to apposed, tethered membranes). SNAREs have a conserved "SNARE domain" with a characteristic heptad repeat. SNAREs are categorized as R-SNAREs if they have a central arginyl residue, or Qa-, Qb-, or Qc-SNAREs with a central glutamyl residue (3). SNAREs form RQaQbQc quaternary cis-or trans-SNARE complexes, which bind SNARE chaperones, including the Sec1/Munc18 family of SNARE binding proteins, and Sec18/NSF (N-ethylmaleimidesensitive factor), an AAA family ATPase that drives SNARE c...

Zippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here, we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 (NSF) catalysis, Sec17 (α-SNAP) either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, Vps33/HOPS promotes productive SNARE assembly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially zipped, Sec17 promotes fusion in either the presence or absence of HOPS, but with faster kinetics when HOPS is absent, suggesting that ejection of the SM is a rate-limiting step.