Fusogenic pairings of vesicle-associated membrane proteins (VAMPs) and plasma membrane t-SNAREs--VAMP5 as the exception.
Fusogenic pairings of vesicle-associated membrane proteins (VAMPs) and plasma membrane t-SNAREs--VAMP5 as the exception.
复制标题
DOI：
10.1371/journal.pone.0014238
复制
发表时间：
2010-12-06
期刊：
影响因子：
3.7
通讯作者：
Hu C
中科院分区：
文献类型：
--
作者：
Hasan N;Corbin D;Hu C
胞内囊泡融合由囊泡（v-SNARE）和靶膜（t-SNARE）上的SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白的相互作用介导。囊泡相关膜蛋白（VAMP）是位于高尔基体后囊泡区室的v-SNARE。为了充分理解每个VAMP在囊泡运输中的具体作用，重要的是确定VAMP是否具有差异性膜融合活性。在这项研究中，我们开发了一种细胞融合试验，通过β-半乳糖苷酶的活化表达来定量SNARE介导的膜融合事件，并检查了7种VAMP之间的融合配对，即，VAMP 1、2、3、4、5、7和8，以及两种质膜t-SNARE复合物，突触融合蛋白1/SNAP-25和突触融合蛋白4/SNAP-25。VAMP 1、2、3、4、7和8有效地驱动融合，而VAMP 5不能介导与t-SNARE的融合。通过在相同水平上表达VAMP 1、3、4、7和8，我们进一步比较了它们的膜融合活性。VAMP 1和3具有相当的和最高的融合活性，而VAMP 4、7和8显示出低30-50%的融合活性。此外，我们确定了细胞融合活性对VAMP 1表达水平的依赖性。依赖性数据分析表明，VAMP蛋白在细胞融合反应中不存在协同性。这些数据表明，VAMP具有不同的膜融合能力，并暗示，除了VAMP 5，VAMP在介导与质膜t-SNARE的融合中基本上是冗余的。
Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities. In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction. These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.
登录
查看更多内容
影响因子：
7.8
作者：
Galli, T;Chilcote, T;Mundigl, O;Binz, T;Niemann, H;De Camilli, P
通讯作者：
De Camilli, P
影响因子：
4.8
作者：
Martin, LB;Shewan, A;James, DE
通讯作者：
James, DE
影响因子：
4.1
作者：
Jagadish, MN;Fernandez, CS;Ward, CW
通讯作者：
Ward, CW
影响因子：
4.8
作者：
Fasshauer, D;Margittai, M
通讯作者：
Margittai, M
影响因子：
64.5
作者：
Kesavan, Jaideep;Borisovska, Maria;Bruns, Dieter
通讯作者：
Bruns, Dieter