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
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DOI：
10.1074/jbc.m700971200
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发表时间：
2007-05-18
影响因子：
4.8
通讯作者：
Wickner, William
中科院分区：
文献类型：
--
作者：
Fratti, Rutilio A.;Collins, Kevin M.;Wickner, William
SNARE蛋白形成4个α -螺旋SNARE结构域的束，在束的“0层”具有保守的极性氨基酸3Q和1R。先前的研究已经证实了3Q（）的重要性。但尚未表明它是否调节SNARE复合体的组装或组装后的SNARE的下游功能。酵母液泡融合需要调节脂质（麦角甾醇、磷酸肌苷和二酰基甘油）、Rab Ypt7p、Rab效应复合物HOPS和4个SNAREs: Q-SNAREs Vti1p、Vam3p、Vam7p和R-SNARE Nyv1p。我们现在报道，Vam7p或Nyv1p的0层Gln或Arg残基的改变分别强烈抑制融合。携带野生型Nyv1p的液泡对野生型Vam7p和Vam7（Q283R）有很好的区分，而携带Nyv1（R191Q）的液泡更偏爱Vam7（Q283R）。旋转0层中精氨酸的位置增加了Vam7p的K-m，但不影响最大熔化速率。Vam7（Q283R）形成稳定的2Q（。）不促进融合的2R复合物。然而，通过亲脂两亲性氯丙嗪或磷脂酶C抑制剂U73122恢复融合，扰乱膜的脂质相。因此，由0层调控的SNARE功能与脂质紧密耦合，脂质必须重新排列才能融合。
SNARE proteins form bundles of four alpha-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 3Q(.)1R 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 2Q(.)2R 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.