Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

Abstract

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...

Co-regulation of theGlycine maxsoluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-containing regulon occurs during defense to a root pathogen

“…The experiments presented here have also examined the relative changes in transcript abundance of SNARE, demonstrating that the genes appear to be co-regulated. The co-regulation of different vesicle components observed here in this system has been seen in other organisms, some of them non-plant systems, and functional genomics screens have revealed this co-regulation can be quite extensive (Shanks et al 2012;Liberali et al 2014;Pant et al 2014Pant et al , 2015aZick et al 2015). However, very little published data is available in plants.…”

Disease resistance through impairment of α-SNAP–NSF interaction and vesicular trafficking by soybean Rhg1

“…In mice, α-SNAP mutations such as the hyh allele are homozygous-lethal, as are NSFnull comatose alleles in Drosophila (33,34). Artificial mutations at the penultimate C-terminal leucine of yeast or animal α-SNAPs no longer stimulate NSF ATPase, impair SNARE recycling, block secretion, and cause apoptosis in cell cultures (14,25,29). What is unusual about soybean Rhg1 is that sabotaging this core housekeeping function contributes to a beneficial trait-a trait that has been widely selected for by soybean breeders in recent decades to help control a disease that annually causes billions of dollars in lost food harvest worldwide.…”

“…No amino acid polymorphisms are predicted in the Glyma.18G022400 or Glyma.18G022700 products from SCN-susceptible as opposed to SCN-resistant Rhg1 haplotypes, but Rhg1 copy-number expansion constitutively elevates the transcript levels of these genes in SCN-resistant plants (9). The polymorphisms in the Rhg1 Glyma.18G022500-encoded α-SNAP are at the highly conserved C terminus, which in mammal and yeast systems directly contacts NSF and is required for activation of SNARE disassembly (8)(9)(10)14).…”

“…S1A) (10). In vitro studies of yeast and animal NSF have demonstrated that this leucine enhances NSF ATPase activity, and that α-SNAP with an engineered leucine-toalanine substitution at this position no longer stimulates ATPase activity or SNARE disassembly (10,14). We therefore assessed the effects of penultimate leucine substitutions in Rhg1 α-SNAPs.…”

“…SNARE complex disassembly by α-SNAP and NSF is essential for vesicular trafficking and, as such, has been studied in considerable detail. X-ray crystallography, singlemolecule fluorescence spectroscopy, and cryo-EM have provided high-resolution structural insights into the dynamics of SNARE-α-SNAP-NSF interactions (12)(13)(14). Multiple α-SNAPs stimulate disassembly of one SNARE bundle in a 20S supercomplex that includes a hexameric ring of six NSF proteins, which couple ATP hydrolysis to force-generating conformational changes.…”