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Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Han B, Tiwari A, Kenworthy AK - Traffic (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.

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Related in: MedlinePlus

The affinity of overexpressed Cav1 for DRMs differs as a function of the tag. DRMs were isolated from (A) untransfected COS-7 cells or cells transiently transfected with (B) Cav1-myc, (C) P132L-myc, (D) Cav1-GFP, (E) P132L-GFP, (F) Cav1-mCherry or (G) P132L-mCherry. Fractions were analyzed by SDS–PAGE/western blotting. The levels of (H) overexpressed Cav1 and (I) endogenous Cav1 in each fraction were quantified by densitometry. The position of endogenous Cav1 in DRMs is indicated with a red line in (A–F). Bars represent mean ± SD for two independent experiments.
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fig10: The affinity of overexpressed Cav1 for DRMs differs as a function of the tag. DRMs were isolated from (A) untransfected COS-7 cells or cells transiently transfected with (B) Cav1-myc, (C) P132L-myc, (D) Cav1-GFP, (E) P132L-GFP, (F) Cav1-mCherry or (G) P132L-mCherry. Fractions were analyzed by SDS–PAGE/western blotting. The levels of (H) overexpressed Cav1 and (I) endogenous Cav1 in each fraction were quantified by densitometry. The position of endogenous Cav1 in DRMs is indicated with a red line in (A–F). Bars represent mean ± SD for two independent experiments.

Mentions: The results of the velocity sucrose gradient centrifugation fractionation analysis indicate that overexpressed Cav1-FPs fail to form 70S complexes properly. To gain further insight into the properties of these various complexes, we asked whether they could associate with detergent-resistant membranes (DRMs), a characteristic feature of Cav1 (60,84,99). We conducted density gradient centrifugation analysis to separate DRM and detergent-soluble membrane. Control experiments in untransfected cells verified that endogenous Cav1 and Cav2 associated with DRMs and that these fractions were distinct from detergent-soluble membranes containing calnexin (Figure 10A). A large fraction of Cav1-myc also associated with DRMs, although the position of this DRM peak was shifted slightly relative to the position of endogenous Cav1 in control cells (Figure 10B,H). In contrast, Cav1-GFP and Cav1-mCherry had only modest affinity for DRMs (Figure 10D,F,H), and P132L constructs were either degraded or essentially completely detergent soluble (Figures 10C,E,G, S4 and S5). These results further emphasize that overexpressed Cav1 and P132L-Cav1 have different fates depending on the type of tag they are fused with.


Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Han B, Tiwari A, Kenworthy AK - Traffic (2015)

The affinity of overexpressed Cav1 for DRMs differs as a function of the tag. DRMs were isolated from (A) untransfected COS-7 cells or cells transiently transfected with (B) Cav1-myc, (C) P132L-myc, (D) Cav1-GFP, (E) P132L-GFP, (F) Cav1-mCherry or (G) P132L-mCherry. Fractions were analyzed by SDS–PAGE/western blotting. The levels of (H) overexpressed Cav1 and (I) endogenous Cav1 in each fraction were quantified by densitometry. The position of endogenous Cav1 in DRMs is indicated with a red line in (A–F). Bars represent mean ± SD for two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440517&req=5

fig10: The affinity of overexpressed Cav1 for DRMs differs as a function of the tag. DRMs were isolated from (A) untransfected COS-7 cells or cells transiently transfected with (B) Cav1-myc, (C) P132L-myc, (D) Cav1-GFP, (E) P132L-GFP, (F) Cav1-mCherry or (G) P132L-mCherry. Fractions were analyzed by SDS–PAGE/western blotting. The levels of (H) overexpressed Cav1 and (I) endogenous Cav1 in each fraction were quantified by densitometry. The position of endogenous Cav1 in DRMs is indicated with a red line in (A–F). Bars represent mean ± SD for two independent experiments.
Mentions: The results of the velocity sucrose gradient centrifugation fractionation analysis indicate that overexpressed Cav1-FPs fail to form 70S complexes properly. To gain further insight into the properties of these various complexes, we asked whether they could associate with detergent-resistant membranes (DRMs), a characteristic feature of Cav1 (60,84,99). We conducted density gradient centrifugation analysis to separate DRM and detergent-soluble membrane. Control experiments in untransfected cells verified that endogenous Cav1 and Cav2 associated with DRMs and that these fractions were distinct from detergent-soluble membranes containing calnexin (Figure 10A). A large fraction of Cav1-myc also associated with DRMs, although the position of this DRM peak was shifted slightly relative to the position of endogenous Cav1 in control cells (Figure 10B,H). In contrast, Cav1-GFP and Cav1-mCherry had only modest affinity for DRMs (Figure 10D,F,H), and P132L constructs were either degraded or essentially completely detergent soluble (Figures 10C,E,G, S4 and S5). These results further emphasize that overexpressed Cav1 and P132L-Cav1 have different fates depending on the type of tag they are fused with.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.

Show MeSH
Related in: MedlinePlus