The structural and functional implications of linked SNARE motifs in SNAP25.
The structural and functional implications of linked SNARE motifs in SNAP25.
复制标题
SNAP25 中连接的 SNARE 基序的结构和功能含义。
DOI：
10.1091/mbc.e08-04-0344
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发表时间：
2008
影响因子：
3.3
通讯作者：
Holz,RonaldW
中科院分区：
文献类型：
--
作者：
Wang,Li;Bittner,MaryA;Axelrod,Daniel;Holz,RonaldW
我们研究了具有两个SNARE基序（SN 1和SN 2）的SNAP 25的功能和结构含义。构建了一种膜结合的分子内FRET探针，用于报道活细胞中N-末端SN 1和C-末端SN 2的折叠。含有任一或两个SNARE基序的膜结合构建体也单独标记供体或受体荧光团。通过形成抗SDS复合物和使用光谱法在体外测量FRET的变化以及使用TIRF显微镜在活细胞质膜中测量FRET的变化来监测探针与其他SNARE的相互作用。探针形成了预测的SDS抗性SNARE复合物。FRET测量结果显示，syntaxin诱导SN 1和SN 2的N-末端的密切关联。这种结合需要SNARE基序位于同一分子中。出乎意料的是，突触融合蛋白诱导的FRET被VAMP阻止。全长SNAP 25构建体和其分离的膜结合组成链的组合都支持已被允许流下的透化嗜铬细胞中的分泌。然而，只有全长SNAP 25构建体能够在耗尽之前从完整细胞或透化细胞稳健分泌。实验表明，双齿结构允许特定的构象与syntaxin和VAMP的复合物和促进功能的SN 1和SN 2的胞吐。
We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.