Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Abstract

SNARE proteins form bundles of four ␣-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q⅐1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7 Q283R , yet Vam7 Q283R is preferred by vacuoles with Nyv1 R191Q . Rotation of the position of the arginine in the 0-layer increases the K m for Vam7p but does not affect the maximal rate of fusion. Vam7 Q283R forms stable 2Q⅐2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion. SNARE 4 proteins (1) are vital for membrane fusion. Initially discovered in neuronal tissues, SNAREs are found on all organelles of the exocytic and endocytic pathways, from yeast to humans. Their characteristic feature is a heptad-repeat "SNARE domain," flanked by varied N-domains and by C-terminal membrane anchors, either a single membrane-spanning apolar polypeptide or an acyl anchor. SNAREs form 4-helical complexes through their SNARE domains (2). Although the residues in each SNARE that face the others in a 4-helical complex are generally apolar, 3 glutamine (Q) and 1 arginine (R) near the center of the 4-complexed SNARE domains form a conserved and polar "0-layer" (3). Recombinant neuronal SNAREs spontaneously assemble into stable bundles, yet SNARE complex assembly in vivo requires SNARE-binding proteins of the Sec1-Munc18 "SM" family. SNARE complexes are disassembled by two chaperones: Sec17p/␣-SNAP, which binds directly to SNARE complexes, and Sec18p/NSF, which binds to Sec17p/␣-SNAP and couples the energy of ATP binding and hydrolysis to SNARE complex disassembly (4). SNARE complexes form in cis, with each SNARE anchored to the same membrane, or in trans, with SNAREs anchored to apposed, "tethered" membranes.SNARE function has been studied in vivo, on isolated organelles, and through the reconstitution of recombinant SNAREs into proteoliposomes. When several SNAREs, which are found in vivo on a "target" membrane, are co-reconstituted into one population of liposomes, they form a t-SNARE complex. These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impress...

Yeast vacuole fusion: A model system for eukaryotic endomembrane dynamics

“…According to several studies, it seems that the Q-SNAREs Vam3, Vam7 and Vti1 on one membrane pair with the R-SNARE Nyv1 from the other membrane. 53,[57][58][59] This arrangement is sufficient to drive liposome fusion using purified SNAREs. 60 The proteins form a zipper-like complex, which propagates from the N-terminal, cytoplasmic side to the transmembrane proximal part of the SNARE domain.…”

“…59 In this case, trans-SNAREs form in the previously described manner and are detectable in the presence of a fusion inhibitor MED (MARCKS effector domain), which binds the regulatory lipid PI(4,5)P 2 . 58,59 Although this assay provides an important step forward, it will be necessary to develop additional approaches that do not depend on any SNARE deletion strain and are capable of dissecting sub-steps in the SNARE assembly.…”

“…95 Comparable to SNARE-mediated fusion at other compartments within eukaryotes, 67 the vacuolar trans-SNARE complex bridging two vacuoles must contain three Q-(Vam3, Vti1, Vam7) and one R-SNARE (Nyv1) in the 0-layer. 58 It also depends on specific lipids, which form distinct domains where SNAREs are assembled. 58 The R-SNARE Ykt6.…”

“…58 It also depends on specific lipids, which form distinct domains where SNAREs are assembled. 58 The R-SNARE Ykt6. Ykt6 is an R-SNARE involved in vacuole fusion that was initially identified as being involved in Golgi transport steps 107 and transport from the ER to the Golgi.…”

In VivoAnalysis of Membrane Fusion

“…However, neither of these two models has been fully supported by in vivo tests Hanson et al, 1997;Lauer et al, 2006;Scales et al, 2001). Although other complexes can be assembled in vitro, only SNAREs containing a QabcR 'QQQR' core complex can efficiently support fusion in vivo (Dilcher et al, 2001;Fratti et al, 2007;Gil et al, 2002;Ossig et al, 2000;Wang et al, 2001;Wei et al, 2000). At each trafficking reaction along the secretory pathway a unique core complex is assembled, leading to the model that the SNAREs provide the specificity of fusion (McNew et al, 2000a;Parlati et al, 2002;Scales et al, 2000;So¨llner et al, 1993b).…”