Han Bing, Tiwari Ajit, Kenworthy Anne K
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.
Traffic. 2015 Apr;16(4):417-38. doi: 10.1111/tra.12254.
Caveolin-1 (Cav1) is the primary scaffolding protein of caveolae, flask-shaped invaginations of the plasma membrane thought to function in endocytosis, mechanotransduction, signaling and lipid homeostasis. A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins. However, how different tags affect the behavior of ectopically expressed Cav1 is still largely unknown. To address this question, we performed a comparative analysis of the subcellular distribution, oligomerization state and detergent resistance of transiently overexpressed Cav1 labeled with three different C-terminal tags (EGFP, mCherry and myc). We show that addition of fluorescent protein tags enhances the aggregation and/or degradation of both wild-type Cav1 and an oligomerization defective P132L mutant. Strikingly, complexes formed by overexpressed Cav1 fusion proteins excluded endogenous Cav1 and Cav2, and the properties of native caveolins were largely preserved even when abnormal aggregates were present in cells. These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae. They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.
小窝蛋白-1（Cav1）是小窝（质膜呈烧瓶状内陷结构，被认为在内吞作用、机械转导、信号传导和脂质稳态中发挥作用）的主要支架蛋白。我们目前关于小窝蛋白和小窝的大量知识来源于对瞬时过表达的、C末端标记的小窝蛋白的研究。然而，不同的标签如何影响异位表达的Cav1的行为在很大程度上仍然未知。为了解决这个问题，我们对用三种不同C末端标签（EGFP、mCherry和myc）标记的瞬时过表达的Cav1的亚细胞分布、寡聚化状态和去污剂抗性进行了比较分析。我们发现，荧光蛋白标签的添加增强了野生型Cav1和寡聚化缺陷型P132L突变体的聚集和/或降解。令人惊讶的是，过表达的Cav1融合蛋白形成的复合物排除了内源性Cav1和Cav2，并且即使细胞中存在异常聚集体，天然小窝蛋白的特性在很大程度上仍得以保留。这些发现表明，标签策略的差异可能是先前发表的关于Cav1研究中变异的一个来源，并且过表达的Cav1可能在小窝之外发挥功能作用。它们还强调了基于标记的Cav1瞬时过表达对当前知识进行批判性重新评估的必要性。
