Selective cell-surface labeling of the molecular motor protein prestin.
Selective cell-surface labeling of the molecular motor protein prestin.
复制标题
分子运动蛋白 prestin 的选择性细胞表面标记。
DOI：
10.1016/j.bbrc.2011.05.121
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发表时间：
2011
影响因子：
3.1
通讯作者：
Raphael,RobertM
中科院分区：
文献类型：
--
作者：
McGuire,RyanM;Silberg,JonathanJ;Pereira,FredA;Raphael,RobertM
普雷斯廷是一种N-和C-末端位于细胞质的多通道跨膜蛋白，必须运输到质膜以实现其作为分子马达的细胞功能。研究活细胞内普雷斯廷序列-功能关系的一个挑战是分离氨基酸取代对普雷斯廷运输、质膜定位和功能的影响。为了开发一种直接评估质膜上普雷斯廷水平的方法，我们已经研究了将普雷斯廷融合到单次跨膜蛋白是否会导致具有可以在活细胞中检测到的表面暴露的N-末端标签的功能性融合蛋白。我们发现，融合的生物素受体肽（BAP）和跨膜结构域的血小板衍生生长因子受体（PDGFR）的N-末端prestin-GFP产生的膜蛋白，可以代谢标记的生物素，贩运到质膜，并选择性地检测在质膜上使用荧光标记的链霉亲和素。此外，我们表明，除了表面可检测的标签和一个单程跨膜结构域的普雷斯廷不破坏其电压敏感的活动。
Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.