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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.

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Colocalization of Cav–KKSL, ADRP, and Nile red staining. Endogenous ADRP (A) and transiently expressed Cav–KKSL (B) in a COS cell were visualized by IF. C, Merged image of A and B. Nile red stain (D) and IF visualization of Cav–KKSL (E) in a transiently transfected FRT cell. F, Merged image of D and E.
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Figure 2: Colocalization of Cav–KKSL, ADRP, and Nile red staining. Endogenous ADRP (A) and transiently expressed Cav–KKSL (B) in a COS cell were visualized by IF. C, Merged image of A and B. Nile red stain (D) and IF visualization of Cav–KKSL (E) in a transiently transfected FRT cell. F, Merged image of D and E.

Mentions: To determine whether the Cav–KKSL-positive structures were lipid storage droplets, Cav–KKSL-expressing FRT cells were double-labeled with antibodies to Cav–KKSL and with either Nile red or with antibodies to ADRP. Nile red stained the lipid droplet cores, whereas both Cav–KKSL and ADRP were localized to the surface of the droplets (Fig. 2). In some transfected cells, almost all Nile-red positive droplets were also positive for Cav–KKSL. In others (Fig. 2 F), some droplets stained only for Nile red.


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)

Colocalization of Cav–KKSL, ADRP, and Nile red staining. Endogenous ADRP (A) and transiently expressed Cav–KKSL (B) in a COS cell were visualized by IF. C, Merged image of A and B. Nile red stain (D) and IF visualization of Cav–KKSL (E) in a transiently transfected FRT cell. F, Merged image of D and E.
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Related In: Results  -  Collection

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

Figure 2: Colocalization of Cav–KKSL, ADRP, and Nile red staining. Endogenous ADRP (A) and transiently expressed Cav–KKSL (B) in a COS cell were visualized by IF. C, Merged image of A and B. Nile red stain (D) and IF visualization of Cav–KKSL (E) in a transiently transfected FRT cell. F, Merged image of D and E.
Mentions: To determine whether the Cav–KKSL-positive structures were lipid storage droplets, Cav–KKSL-expressing FRT cells were double-labeled with antibodies to Cav–KKSL and with either Nile red or with antibodies to ADRP. Nile red stained the lipid droplet cores, whereas both Cav–KKSL and ADRP were localized to the surface of the droplets (Fig. 2). In some transfected cells, almost all Nile-red positive droplets were also positive for Cav–KKSL. In others (Fig. 2 F), some droplets stained only for Nile red.

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