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Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets.

Ostermeyer AG, Paci JM, Zeng Y, Lublin DM, Munro S, Brown DA - J. Cell Biol. (2001)

Bottom Line: We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP.Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets.Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.

ABSTRACT
Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.

Show MeSH
Localization of Cav–ΔN2 and caveolin-2. IF localization of Cav–ΔN2 (A) and caveolin-2 (B) in transiently transfected FRT cells. The Golgi apparatus was overexposed to show the droplets.
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Figure 3: Localization of Cav–ΔN2 and caveolin-2. IF localization of Cav–ΔN2 (A) and caveolin-2 (B) in transiently transfected FRT cells. The Golgi apparatus was overexposed to show the droplets.

Mentions: We also examined the localization of Cav–ΔN2, a mutant made for unrelated studies, that lacks residues 46–95 of caveolin-1, including almost the entire downstream half of the NH2-terminal hydrophilic domain. In some cells, Cav–ΔN2 was present in the Golgi apparatus (identified as a perinuclear structure labeled with FITC-lens lectin, not shown). However, in most cells the protein was present in lipid droplets, identified by their characteristic morphology. Both patterns are evident in the cell shown in Fig. 3 A. The presence of Cav–ΔN2 in lipid droplets showed that the KKSL tag is not required for the accumulation of Cav–KKSL in the droplets. We also conclude that residues 46–95, which include most of the caveolin scaffolding domain previously reported to mediate binding of caveolin-1 to other proteins (Smart et al. 1999), are not required for lipid droplet targeting.


Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets.

Ostermeyer AG, Paci JM, Zeng Y, Lublin DM, Munro S, Brown DA - J. Cell Biol. (2001)

Localization of Cav–ΔN2 and caveolin-2. IF localization of Cav–ΔN2 (A) and caveolin-2 (B) in transiently transfected FRT cells. The Golgi apparatus was overexposed to show the droplets.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198801&req=5

Figure 3: Localization of Cav–ΔN2 and caveolin-2. IF localization of Cav–ΔN2 (A) and caveolin-2 (B) in transiently transfected FRT cells. The Golgi apparatus was overexposed to show the droplets.
Mentions: We also examined the localization of Cav–ΔN2, a mutant made for unrelated studies, that lacks residues 46–95 of caveolin-1, including almost the entire downstream half of the NH2-terminal hydrophilic domain. In some cells, Cav–ΔN2 was present in the Golgi apparatus (identified as a perinuclear structure labeled with FITC-lens lectin, not shown). However, in most cells the protein was present in lipid droplets, identified by their characteristic morphology. Both patterns are evident in the cell shown in Fig. 3 A. The presence of Cav–ΔN2 in lipid droplets showed that the KKSL tag is not required for the accumulation of Cav–KKSL in the droplets. We also conclude that residues 46–95, which include most of the caveolin scaffolding domain previously reported to mediate binding of caveolin-1 to other proteins (Smart et al. 1999), are not required for lipid droplet targeting.

Bottom Line: We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP.Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets.Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.

ABSTRACT
Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.

Show MeSH