Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion
Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion
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DOI：
10.1083/jcb.145.7.1435
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发表时间：
1999-06-28
影响因子：
7.8
通讯作者：
Wickner, W
中科院分区：
文献类型：
--
作者：
Ungermann, C;von Mollard, GF;Wickner, W
液泡 SNARE，包括 t-SNARE Vam3p 和 Vam7p 以及 V-SNARE Nyv1p，存在于分离细胞器的多亚基“顺式”复合体中。我们现在通过质谱法鉴定出 v-SNARE Vti1p 和 Ykt6p 作为免疫隔离的液泡 SNARE 复合物的附加成分。用抗-Vti1p对洗涤剂提取物进行免疫耗竭，去除与Vam3p复合物中的所有Ykt6p，用抗-Ykt6p免疫耗竭去除与Vam3p复合的所有Vti1p，用抗-Nyv1p免疫耗竭去除与其他SNARE复合物中的所有Ykt6p，证明它们都在同一个顺式多SNARE复合物中。在引发（分解顺式 SNARE 复合物）后，五种 SNARE 蛋白中任何一种的抗体仍然会抑制融合测定，直到对接阶段完成，这表明每个 SNARE 在对接中都发挥着作用。此外，vti1 温度敏感等位基因在我们的反应中导致合成融合缺陷表型。我们的数据表明，液泡-液泡融合需要一个由五个 SNARE 组成的 cis-SNARE 复合体，即 t-SNARE Vam3p 和 Vam7p 以及 v-SNARE Nyv1p、Vti1p 和 Ykt6p。
Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the V-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.