title: Tagging strategies strongly affect the fate of overexpressed caveolin-1.
abstract: 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.
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors: Bing
authors: B
authors: Han
authors: Ajit
authors: A
authors: Tiwari
authors: Anne K
authors: AK
authors: Kenworthy
mesh_terms: D000818:Animals; D019556:COS Cells; D051242:Caveolin 1 / Q000378:metabolism*; D051243:Caveolin 2 / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D051242:Caveolin 1; D051243:Caveolin 2; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D016271:Proto-Oncogene Proteins c-myc; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein
keywords: blue native gel electrophoresis; breast cancer; caveolae; caveolin; detergent-resistant membranes; fluorescent proteins; oligomerization; overexpression; velocity gradient centrifugation
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