Tagging strategies strongly affect the fate of overexpressed caveolin-1.
Bottom Line: A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins.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.
Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.Show MeSH
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Mentions: BN-PAGE separates membrane protein complexes using mild conditions that preserve protein–protein interactions (93). We combined the use of BN-PAGE and dual-color detection of immunoblotted proteins so that we could simultaneously detect two proteins on one membrane. To determine optimal solubilization conditions for these experiments, we compared several different detergents, including 0.5% Triton-X-100, 60 mm octylglucoside, 1% digitonin and 1% n-dodecyl β-D-maltoside (DDM), detergents commonly used for BN-PAGE analyses (87,91,92,94), where 0.5% Triton-X-100 was unable to solubilize Cav1 effectively. However, for all the other conditions tested, endogenous Cav1 was isolated as part of a high molecular weight complex from COS-7 cells (Figure 2). For example, in digitonin-solubilized cells, Cav1 migrates as part of a ∼600 kDa complex. The is very similar in size to previous reports that solubilized Cav1 with octylglucoside (87) or DDM (92) and likely corresponds to the core Cav1 8S unit complexes observed by the velocity gradient fractionation (60). Cav1 is known to form hetero-oligomers with Cav2 (95,96), so we also tested for the presence of Cav2 in these complexes. As expected, endogenous Cav2 perfectly co-migrated with Cav1. Because digitonin solubilization yielded the best resolution of complexes, this condition was chosen for use for further studies.
Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.