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. 2007 Dec 12;26(24):4935-45.
doi: 10.1038/sj.emboj.7601915. Epub 2007 Nov 15.

Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion

Youngsoo Jun  1 , Hao Xu, Naomi Thorngren, William Wickner
Affiliations

Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion

Youngsoo Jun et al. EMBO J. .

Abstract

Intracellular membrane fusion requires SNARE proteins in a trans-complex, anchored to apposed membranes. Proteoliposome studies have suggested that SNAREs drive fusion by stressing the lipid bilayer via their transmembrane domains (TMDs), and that SNARE complexes require a TMD in each docked membrane to promote fusion. Yeast vacuole fusion is believed to require three Q-SNAREs from one vacuole and the R-SNARE Nyv1p from its fusion partner. In accord with this model, we find that fusion is abolished when the TMD of Nyv1p is replaced by lipid anchors, even though lipid-anchored Nyv1p assembles into trans-SNARE complexes. However, normal fusion is restored by the addition of both Sec18p and the soluble SNARE Vam7p. In restoring fusion, Sec18p promotes the disassembly of trans-SNARE complexes, and Vam7p enhances their assembly. Thus, either the TMD of this R-SNARE is not essential for fusion, and TMD-mediated membrane stress is not the only mode of trans-SNARE complex action, or these SNAREs have more flexibility than heretofore appreciated to form alternate functional complexes that violate the 3Q:1R rule.

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Figures

Figure 1
Figure 1
TEV protease-mediated Nyv1p inactivation prevents homotypic yeast vacuole fusion in vitro. (A) Schematic representation of wild-type Nyv1p and Nyv1p-TEV. (B) TEV protease specifically inactivates Nyv1p-TEV vacuoles for fusion. BJ3505 (NYV1) and DKY6281 (NYV1) vacuoles or BJ3505 NYV1-TEV and DKY6281 NYV1-TEV vacuoles were incubated at 27°C in fusion reactions (see Materials and methods) in the presence of indicated concentrations of TEV protease. After 90 min, a portion was assayed for fusion (B), and the rest centrifuged to sediment vacuoles, which were resuspended in SDS sample buffer and analyzed by SDS–PAGE and immunoblotting (C). (C) Fusion inactivation by TEV protease correlates with the removal of Nyv1p-TEV from vacuoles. The anti-Pep4p blot serves as a loading control. Data are mean±s.e.m. (n=3).
Figure 2
Figure 2
The Nyv1p N-terminal longin domain is not essential for vacuole fusion. (A) Nyv1p and its derivatives. (B) Nyv1p longin domain deletions support fusion. BJ3505 NYV1 and DKY6281 NYV1 vacuoles, BJ3505 NYV1-Δ(2–99) and DKY6281 NYV1-Δ(2–99) vacuoles, or BJ3505 NYV1-Δ(2–160) and DKY6281 NYV1-Δ(2–160) vacuoles were incubated at 27°C in the absence or presence of antibodies to Vam3p, antibodies to Nyv1p, or recombinant Vam7p and, after 90 min, assayed for fusion. Data are mean±s.e.m. (n=3). (C) Vacuoles from BJ3505 NYV1, BJ3505 NYV1-Δ(2–99), BJ3505 NYV1-Δ(2–160), DKY6281 NYV1, DKY6281 NYV1-Δ(2–99), and DKY6281 NYV1-Δ(2–160) were analyzed by SDS–PAGE and immunoblotting.
Figure 3
Figure 3
Fusion is restored to vacuoles that lack the TMD of Nyv1p by added Sec18p and Vam7p. (A) Wild-type Nyv1p and Nyv1p-CCIIM. (B) Protein profiles of NYV1, nyv1Δ, and NYV1-CCIIM vacuoles. Vacuoles purified from BJ3505 NYV1, BJ3505 NYV1-CCIIM, BJ3505 nyv1Δ, DKY6281 NYV1, and DKY6281 NYV1-CCIIM were analyzed by SDS–PAGE and immunoblotting. (C) Triton X-114 phase partitioning analysis of the hydrophobicity of wild-type Nyv1p with its TMD, Nyv1p-CCIIM, and the cytoplasmic domain of Nyv1p without an apolar anchor was done with primed BJ3505 NYV1 vacuoles, BJ3505 NYV1-CCIIM vacuoles, and BJ3505 nyv1Δ vacuoles supplemented with recombinant GST-sNyv1p, as described (Bordier, 1981). (D) The addition of both Sec18p and Vam7p enables Nyv1p-CCIIM vacuoles to fuse. Wild-type or Nyv1p-CCIIM vacuoles were incubated on ice or at 27°C in fusion reactions with indicated proteins, added from the start of the incubation. After 90 min, reactions were assayed for fusion. Data are mean±s.e.m. (n=3).
Figure 4
Figure 4
Nyv1p-CCIIM engages in vacuole fusion. (A) The Sec18p/Vam7p-mediated fusion of Nyv1p-CCIIM vacuoles is inhibited by antibodies to Nyv1p, relieved only by recombinant sNyv1p. Vacuoles from BJ3505 NYV1-CCIIM and DKY6281 NYV1-CCIIM were incubated in fusion reactions containing both Sec18p and Vam7p at 27°C in the presence of indicated proteins. After 90 min, reactions were assayed for fusion. Data represent mean±s.e.m. (n=3). To optimize the chance of seeing relief from αNyv1p inhibition, we employed 3.3 μM GST-Vam7p (lanes 8 and 13), a level which itself often causes some fusion inhibition (lane 13). (B) The zero-layer arginine of Nyv1p-CCIIM is important for Nyv1p-CCIIM vacuole fusion. Standard fusion assays (27°C, 90 min) bore BJ3505 nyv1Δ vacuoles and either DKY6281 NYV1-CCIIM or DKY6281 NYV1-CCIIM R192Q vacuoles. Vam7p (638 nM), his6-Sec18p (63.8 nM), and anti-Vam3p (444 nM) were added where indicated.
Figure 5
Figure 5
BJ3505 nyv1Δ vacuoles can fuse with DKY6281 NYV1-CCIIM vacuoles upon addition of Sec18p and Vam7p. Schematic representation of fusion between BJ3505 NYV1-CCIIM vacuoles and DKY6281 NYV1-CCIIM vacuoles (A) or BJ3505 nyv1Δ vacuoles and DKY6281 NYV1-CCIIM vacuoles (B). The dotted line indicates a hypothetical interaction between trans-SNARE complexes. (C) Vacuoles from BJ3505 nyv1Δ and DKY6281 NYV1-CCIIM were incubated for 90 min in fusion reactions on ice or at 27°C with indicated proteins and assayed for fusion. Data are mean±s.e.m. (n=3).
Figure 6
Figure 6
Nyv1p-CCIIM-mediated vacuole fusion requires Sec18p remodeling of trans-SNARE complexes. (A) Assay of trans-SNARE complexes. (B–E) Remodeling of trans-SNARE complexes is required for Nyv1p-CCIIM to support fusion. BJ3505 nyv1Δ and DKY6281 VAM3(ΔN) NYV1-CCIIM vacuoles were incubated in fusion reactions on ice or at 27°C with indicated reagents. After 45 min, aliquots were assayed for fusion (B) and trans-SNARE complexes (C). (D, E) The kinetics of trans-SNARE complex assembly and disassembly. BJ3505 nyv1Δ and DKY6281 VAM3(ΔN) NYV1-CCIIM vacuoles (396 μg each) were mixed with standard reaction buffer and ATP in 3.96 ml. At indicated times, portions (330 μl) were transferred to tubes with Sec18p, Gyp1-46p/Gdi1p, or control buffer before continuing incubation, either on ice or at 27°C. Reactions were stopped by transfer to ice. Aliquots (30 μl) were removed to measure fusion. The remaining 300 μl was mixed with 6 μl of 0.5 mM EDTA, then assayed for trans-SNARE complexes as described in Materials and methods, with proportionate reduction in solubilization buffer from 600 to 400 μl. Incubations were either on ice (lane 1) or at 27°C, with Sec18p added from the start of the incubation (lanes 3 and 10) or after 30 min at 27°C (lanes 8, 9 and 12), and with Gyp1-46p and Gdi1p added from the start of incubation (lane 2). Incubations at 27°C were for 15, 30, 45, or 60 min (lanes 4–7, respectively). After Sec18p addition, the samples in lanes 8 and 9 were incubated for an additional 15 or 30 min at 27°C, respectively. Samples in lanes 10–12 had MED from the start of the incubation; the sample in lane 12 received Sec18p after 30 min of incubation and was then incubated for a further 15 min before analysis.
Figure 7
Figure 7
Vacuoles bearing Nyv1p-CCIIM require additional Sec18p and Vam7p for lipid mixing. A portion of BJ3505 NYV1-CCIIM vacuoles were labeled with self-quenching levels of octadecyl rhodamine B (R18), reisolated, and incubated with an unlabeled portion of these vacuoles under standard fusion conditions to assay for lipid mixing-induced dequenching, as described (Jun and Wickner, 2007). Measurements were taken every 2 min for 90 min, yielding fluorescence values at the onset (F0) and during the reaction (Ft). The final 10 measurements of a sample containing 0.33% (v/v) Triton X-100 were averaged and used as a value for the fluorescence after infinite dilution (FTX100). The relative total fluorescence change ΔFt/FTX100=(Ft−F0)/FTX100 was calculated.
Figure 8
Figure 8
Vam7p restores fusion to vacuoles from BJ3505 VAM3-CCIIM and DKY6281 VAM3-CCIIM. Standard fusion assays (90 min, 27°C) were supplemented with Vam7p and/or 32 nM Sec18p. Anti-Vam3p IgG (888 nM) was added with 365 nM Vam7p and 32 nM Sec18p where indicated. The assay value for a sample held on ice was subtracted from all points except the anti-Vam3p and ice points. Error bars represent standard deviations from three independent experiments. The error bars for the anti-Vam3p and the ice points are covered by their symbols.
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