The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.
The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.
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DOI：
10.3791/50867
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发表时间：
2013-12-13
期刊：
影响因子：
--
通讯作者：
Swiatecka-Urban A
中科院分区：
文献类型：
--
作者：
Cihil KM;Swiatecka-Urban A
膜运输包括蛋白质从质膜运输到细胞内部（即内吞作用），然后运输到溶酶体降解或到质膜再循环。基于细胞的l -谷胱甘肽保护试验可用于研究细胞表面的蛋白质受体、通道、转运体和粘附分子的内吞作用和再循环。内吞试验需要在4ºC下用含有二硫键和n -羟基琥珀酰亚胺（NHS）酯的细胞膜不渗透生物素标记细胞表面蛋白质-在该温度下膜运输不会发生。37℃孵育诱导生物素化质膜蛋白的内吞作用。接下来，再次将温度降低到4℃，以阻止内吞运输，并且生物素中与保留在质膜上的蛋白质共价连接的二硫键被l -谷胱甘肽还原。在这一点上，只有被内吞的蛋白质仍然受到l -谷胱甘肽的保护，从而保持生物素化。细胞裂解后，用链霉亲和素琼脂糖分离生物素化蛋白，从琼脂糖中洗脱，用western blotting检测感兴趣的生物素化蛋白。在循环实验中，生物素化后的细胞在37°C下孵育，用生物素化的蛋白质装载内吞囊泡，生物素中的二硫键共价地附着在质膜上的蛋白质上，与内吞实验中一样，在4°C下用l -谷胱甘肽还原。接下来，细胞在37°C下再次孵育，以允许来自内吞囊泡的生物素化蛋白质循环到质膜。然后将细胞在4℃下孵育，生物素中的二硫键连接到蛋白质上，再循环到质膜上，用l -谷胱甘肽还原。受l -谷胱甘肽保护的生物素化蛋白是那些没有再循环到质膜的蛋白。
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
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影响因子：
5.5
作者：
Swiatecka-Urban, A;Moreau-Marquis, S;Stanton, BA
通讯作者：
Stanton, BA
影响因子：
4.8
作者：
Swiatecka-Urban, A;Duhaime, M;Stanton, BA
通讯作者：
Stanton, BA
影响因子：
5.5
作者：
Le, TL;Joseph, SR;Stow, JL
通讯作者：
Stow, JL
影响因子：
4.8
作者：
Ye, Siying;Cihil, Kristine;Swiatecka-Urban, Agnieszka
通讯作者：
Swiatecka-Urban, Agnieszka
影响因子：
11.4
作者：
BRETSCHER, MS;LUTTER, R
通讯作者：
LUTTER, R