Gabriel Luke, Stevens Zachary, Melikian Haley
University of Massachusetts Medical School, USA.
J Vis Exp. 2009 Dec 23(34):1669. doi: 10.3791/1669.
Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps. Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling. Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression. The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins. In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured. For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands. However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate. In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate. This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.
质膜蛋白是一大类多样的蛋白质，由受体、离子通道、转运体和泵组成。这些蛋白质的活性负责各种关键的细胞事件，包括营养物质输送、细胞兴奋性和化学信号传导。许多质膜蛋白通过内吞运输进行动态调节，内吞运输通过改变蛋白质表面表达来调节蛋白质功能。促进蛋白质内吞作用的机制很复杂，许多膜蛋白的机制尚未完全了解。为了充分理解控制给定蛋白质内吞运输的机制，精确测量该蛋白质的内吞速率至关重要。对于许多受体，直接内吞速率测量通常利用标记的受体配体来实现。然而，对于许多类膜蛋白，如转运体、泵和离子通道，没有可用于测量内吞速率的便捷配体。在本报告中，我们描述了一种用于测量多巴胺转运体（DAT）内吞速率的可逆生物素化方法。该方法为测量内化速率提供了一种直接的方法，并且可以很容易地用于大多数膜蛋白的运输研究。
