Tagging strategies strongly affect the fate of overexpressed caveolin-1
| Autor: | Anne K. Kenworthy, Bing Han, Ajit Tiwari |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Scaffold protein
fluorescent proteins Caveolin 2 Caveolin 1 Green Fluorescent Proteins Biology Endocytosis Biochemistry Mechanotransduction Cellular Cell Line oligomerization Proto-Oncogene Proteins c-myc 03 medical and health sciences 0302 clinical medicine breast cancer Structural Biology Caveolae Cell Line Tumor Caveolin Chlorocebus aethiops Genetics Animals Humans detergent-resistant membranes Molecular Biology 030304 developmental biology 0303 health sciences velocity gradient centrifugation Cell Membrane Cell Biology blue native gel electrophoresis Fusion protein Cell biology Luminescent Proteins 030220 oncology & carcinogenesis COS Cells caveolae caveolin Traffic Interchange mCherry HeLa Cells overexpression |
| Zdroj: | Traffic (Copenhagen, Denmark) |
| ISSN: | 1600-0854 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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