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: A large fraction of the Cav1-FPs and Cav1-myc was found in high molecular weight aggregates in the velocity gradient centrifugation experiments (Figure 8). To further investigate the nature of these aggregates, prior to the velocity gradient centrifugation, we lysed the cells with a combined detergent solution containing 0.2% Triton-X-100 and 0.4% SDS previously shown to disassemble the 70S complexes (60). Under these conditions, Cav1-myc and Cav1-mCherry disassembled into 8S-like complexes (Figure 9A, B, E, F), whereas for Cav1-GFP, a combination of monomers or small oligomers and 8S-like complexes was observed (Figure 9C,D). The GFP tag may, thus, partially interfere with the formation of 8S-like oligomers. Consistent with previous findings that P132L tends to oligomerize poorly (60,84), the P132L constructs dissociated into low molecular weight oligomers (Figures 9G–J and S4B). These findings suggest that although both wild-type and P132L form irregular aggregates and high molecular weight oligomers, the aggregates are formed from different building blocks.
Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.