Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.
Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.
复制标题
通过异位靶向确定囊泡束缚中外囊的功能。
DOI：
10.1007/978-1-0716-2209-4_6
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Guo,Wei
中科院分区：
文献类型：
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作者：
Mei,Kunrong;Liu,Di-Ao;Guo,Wei
我们描述了一个实验，其中异位靶向胞囊亚基Sec3到线粒体被用来确定其在将后高尔基囊泡系在质膜上的作用。在实验中，我们使用一种编码线粒体蛋白Tom20和Sec3 n -末端标记有荧光蛋白mCherry的融合蛋白的质粒，并在酵母细胞中与线粒体标记蛋白CIT1-GFP共表达质粒。然后，我们使用荧光显微镜检测Sec3和CIT1与线粒体上的其他囊胞亚基（如Sec5）之间的共定位。我们进一步检测了Sec3和Sec4之间的共定位，Sec3和Sec4是Rab蛋白和后高尔基泡标记物。通过本实验，我们提出了囊泡亚基Sec3将其他囊泡亚基和分泌囊泡招募到靶膜上，这表明它在囊泡系聚中起关键作用。这种方法可能适用于研究其他拴系复合物的特定运输阶段，也可用于其他真核细胞，如培养的哺乳动物细胞。
We describe an assay, in which ectopically targeting the exocyst subunit Sec3 to mitochondria is used to determine its role in tethering of post-Golgi vesicles to the plasma membrane. In the assay, we use a plasmid that encodes a fusion protein of the mitochondria protein Tom20 and Sec3 N-terminally tagged with the florescence protein mCherry, and coexpress the plasmid in yeast cells with CIT1-GFP, a marker protein of mitochondria. We then detect the colocalization between Sec3 and CIT1 and other exocyst subunits such as Sec5 on mitochondria using fluorescence microscopy. We further detect the colocalization between Sec3 and Sec4, a Rab protein and a marker of post-Golgi vesicles. Through this assay, we propose that the exocyst subunit Sec3 recruits the other exocyst subunits and secretory vesicles to a target membrane, suggesting that it plays a pivotal role in vesicle tethering. This approach is likely appropriate for studying other tethering complexes at their specific stages of trafficking and may also be used in other eukaryotic cells such as the cultured mammalian cells.