Testing the 3Q:1R “Rule”: Mutational Analysis of the Ionic “Zero” Layer in the Yeast Exocytic SNARE Complex Reveals No Requirement for Arginine

Katz, Luba; Brennwald, Patrick

doi:10.1091/mbc.11.11.3849

Cited by 66 publications

(59 citation statements)

References 35 publications

(47 reference statements)

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“…Vacuoles bearing Nyv1p R192Q can fuse when given either Vam7p Q283R , restoring the 3Q⅐1R composition of the 0-layer, or wild-type Vam7p, yielding a 4Q 0-layer. These findings are in accord with studies showing that 4Q SNARE complexes are functional for exocytosis (46) or endoplasmic reticulum to Golgi traffic (45), whereas 2Q⅐2R complexes are at least somewhat defective (45). Studies with recombinant neuronal SNAREs have shown that the syntaxin 0-layer Gln is essential for NSF factor and ␣-SNAP-mediated SNARE bundle disassembly (44), and this may account for some of the loss of fusion that accompanies mutation of the Vam3p 0-layer Gln to Arg in an earlier study (47).…”

Section: Discussionsupporting

confidence: 92%

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

Fratti¹,

Collins²,

Hickey

et al. 2007

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 92%

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

Fratti¹,

Collins²,

Hickey

et al. 2007

Journal of Biological Chemistry

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“…The "four Q configuration" in the zero layer of the SNARE motif is functional The lack of a major phenotype in synaptic exocytosis produced by the Q/R substitution agrees well with previous studies in yeast showing that similar substitutions did not abolish SNARE function (Katz and Brennwald, 2000;Ossig et al, 2000). Previous experiments in yeast, however, did not have the temporal resolution of synaptic exocytosis to detect changes in the kinetics or Figure 6.…”

Section: Substitution Of Synaptobrevin Function By Cellubrevinsupporting

confidence: 87%

Structural Determinants of Synaptobrevin 2 Function in Synaptic Vesicle Fusion

Deák

Shin

Kavalali

et al. 2006

Journal of Neuroscience

136

140

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Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca 2ϩ -evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca 2ϩ -triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.

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“…However, all Q-SNARE complexes composed either of a mutated Snc v-SNARE and one molecule each of Sso and Sec9 (14,15) or of two molecules of Sso and one of Sec9 (16) have also been shown to be functional and to confer exocytosis. In addition, Sec1, a conserved regulator of the Sso/syntaxin t-SNAREs, is known to bind to the assembled SNARE complex and may play a definitive role in facilitating assembly (17,18).…”

mentioning

confidence: 99%

Mso1 Is a Novel Component of the Yeast Exocytic SNARE Complex

Castillo-Flores

Weinberger

Robinson

et al. 2005

Journal of Biological Chemistry

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The yeast exocytic SNARE complex consists of one molecule each of the Sso1/2 target SNAREs, Snc1/2 vesicular SNAREs, and the Sec9 target SNARE, which form a fusion complex that is conserved in evolution. Another protein, Sec1, binds to the SNARE complex to facilitate assembly. We show that Mso1, a Sec1-interacting protein, also binds to the SNARE complex and plays a role in mediating Sec1 functions. Like Sec1, Mso1 bound to SNAREs in cells containing SNARE complexes (i.e. wild-type, sec1-1, and sec18-1 cells), but not in cells in which complex formation is inhibited (i.e. sec4-8 cells). Nevertheless, Mso1 remained associated with Sec1 even in sec4-8 cells, indicating that they act as a pair. Mso1 localized primarily to the plasma membrane of the bud when SNARE complex formation was not impaired but was mostly in the cytoplasm when assembly was prevented. Genetic studies suggest that Mso1 enhances Sec1 function while attenuating Sec4 GTPase function. This dual action may impart temporal regulation between Sec4 turnoff and Sec1-mediated SNARE assembly. Notably, a small region at the C terminus of Mso1 is conserved in the mammalian Munc13/Mint proteins and is necessary for proper membrane localization. Overexpression of Mso1 lacking this domain (Mso1-(1-193)) inhibited the growth of cells bearing an attenuated Sec4 GTPase. These results suggest that Mso1 is a component of the exocytic SNARE complex and a possible ortholog of the Munc13/Mint proteins.The membrane fusion apparatus in eukaryotes relies upon SNAREs 2 to mediate intracellular membrane docking and fusion events. SNAREs are membrane-associated proteins bearing ␣-helical domains (termed SNARE motifs) that can assemble into intermolecular four-helix bundles (1-3). Assembly of the SNARE complex, which is composed of vesicular (v) and target (t) SNAREs (or R-and Q-SNAREs, depending upon the nomenclature followed), bridges apposed bilayers and allows for the extrusion of interposed water molecules, resulting in membrane fusion. Although shown to be sufficient to confer membrane fusion in vitro or under artificial conditions (4 -6), a number of other molecules (termed SNARE regulators) act upstream of the SNAREs and play essential roles in the temporal and spatial control of SNARE assembly in vivo (7-9). At the level of exocytosis, these include the Rab family of small GTPases; the vesicle-tethering exocyst complex; the Sec1/ Munc18 family of SNARE assembly factors, which are conserved from yeast to mammals; and the complexin, Mint, Munc13, and synaptotagmin families of SNARE regulators, which appear to act only upon stimulus-coupled exocytic processes (7-10).In yeast, the exocytic SNARE complex consists of one representative molecule each of the Sso1/2 t-SNAREs (Q-SNARE) (11) and Snc1/2 v-SNAREs (R-SNARE) (12) as well as one molecule of the Sec9 t-SNARE (Q-SNARE) (13). However, all Q-SNARE complexes composed either of a mutated Snc v-SNARE and one molecule each of Sso and Sec9 (14, 15) or of two molecules of Sso and one of Sec9 (16) have also been shown to be ...

show abstract

Testing the 3Q:1R “Rule”: Mutational Analysis of the Ionic “Zero” Layer in the Yeast Exocytic SNARE Complex Reveals No Requirement for Arginine

Cited by 66 publications

References 35 publications

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

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

Structural Determinants of Synaptobrevin 2 Function in Synaptic Vesicle Fusion

Mso1 Is a Novel Component of the Yeast Exocytic SNARE Complex

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