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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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Effect of Cav–KKSL on lipid droplets. A, Lipids from various cell types were separated by HP-TLC and visualized by charring. Lanes 1 and 2 are from a separate plate. Positions of CE, TG, and unidentified lipid X are indicated. Cells used and (percent of cells expressing Cav–KKSL); 1, G418-resistant FRT cells; 2, FRT clone B4 stably expressing Cav–KKSL (19%); 3, transient FRT/vector; 4, transient FRT/Cav–KKSL (32%); 5, COS/vector; 6, COS/Cav–KKSL (36%); 7, HEK 293/vector; 8, HEK 293/Cav–KKSL (53%). Only 10% of the COS cell extracts (lanes 5 and 6) were loaded on the plate. B, Untransfected FRT cells were stained with Nile red. C, COS cells were transiently transfected with Cav–KKSL, detecting the protein by IF (left), or were stained with Nile red (right; a cell with large droplets).
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Figure 7: Effect of Cav–KKSL on lipid droplets. A, Lipids from various cell types were separated by HP-TLC and visualized by charring. Lanes 1 and 2 are from a separate plate. Positions of CE, TG, and unidentified lipid X are indicated. Cells used and (percent of cells expressing Cav–KKSL); 1, G418-resistant FRT cells; 2, FRT clone B4 stably expressing Cav–KKSL (19%); 3, transient FRT/vector; 4, transient FRT/Cav–KKSL (32%); 5, COS/vector; 6, COS/Cav–KKSL (36%); 7, HEK 293/vector; 8, HEK 293/Cav–KKSL (53%). Only 10% of the COS cell extracts (lanes 5 and 6) were loaded on the plate. B, Untransfected FRT cells were stained with Nile red. C, COS cells were transiently transfected with Cav–KKSL, detecting the protein by IF (left), or were stained with Nile red (right; a cell with large droplets).

Mentions: In Fig. 6 A, lipids in confluent cell monolayers in 35-mm dishes were extracted as reported previously (Brown and Rose 1992). In Fig. 7 A, cells were seeded in parallel 60-mm dishes (for lipid extraction) and 35-mm dishes with coverslips (for IF, staining for Cav–KKSL and counterstaining with DAPI to determine the percent of cells that were transfected). For transient transfections, the same transfection mixture was used for both dishes. Nonpolar lipids from cells in confluent 60-mm dishes were extracted with hexane/isopropyl alcohol 3:2 (Underwood et al. 1998). In Fig. 6 A and 7 A, extracted lipids (100% of the FRT and HEK 293 extracts or 10% of the COS extracts) were separated by high-performance thin-layer chromatography (HP-TLC) using hexane/isopropyl ether/acetic acid 65:35:2, and visualized by charring and quantitated by densitometric scanning and comparison to standards on the same plate as described (Brown and Rose 1992). Cholesterol, not expected to accumulate in lipid droplets, was also scanned as an internal control.


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)

Effect of Cav–KKSL on lipid droplets. A, Lipids from various cell types were separated by HP-TLC and visualized by charring. Lanes 1 and 2 are from a separate plate. Positions of CE, TG, and unidentified lipid X are indicated. Cells used and (percent of cells expressing Cav–KKSL); 1, G418-resistant FRT cells; 2, FRT clone B4 stably expressing Cav–KKSL (19%); 3, transient FRT/vector; 4, transient FRT/Cav–KKSL (32%); 5, COS/vector; 6, COS/Cav–KKSL (36%); 7, HEK 293/vector; 8, HEK 293/Cav–KKSL (53%). Only 10% of the COS cell extracts (lanes 5 and 6) were loaded on the plate. B, Untransfected FRT cells were stained with Nile red. C, COS cells were transiently transfected with Cav–KKSL, detecting the protein by IF (left), or were stained with Nile red (right; a cell with large droplets).
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Figure 7: Effect of Cav–KKSL on lipid droplets. A, Lipids from various cell types were separated by HP-TLC and visualized by charring. Lanes 1 and 2 are from a separate plate. Positions of CE, TG, and unidentified lipid X are indicated. Cells used and (percent of cells expressing Cav–KKSL); 1, G418-resistant FRT cells; 2, FRT clone B4 stably expressing Cav–KKSL (19%); 3, transient FRT/vector; 4, transient FRT/Cav–KKSL (32%); 5, COS/vector; 6, COS/Cav–KKSL (36%); 7, HEK 293/vector; 8, HEK 293/Cav–KKSL (53%). Only 10% of the COS cell extracts (lanes 5 and 6) were loaded on the plate. B, Untransfected FRT cells were stained with Nile red. C, COS cells were transiently transfected with Cav–KKSL, detecting the protein by IF (left), or were stained with Nile red (right; a cell with large droplets).
Mentions: In Fig. 6 A, lipids in confluent cell monolayers in 35-mm dishes were extracted as reported previously (Brown and Rose 1992). In Fig. 7 A, cells were seeded in parallel 60-mm dishes (for lipid extraction) and 35-mm dishes with coverslips (for IF, staining for Cav–KKSL and counterstaining with DAPI to determine the percent of cells that were transfected). For transient transfections, the same transfection mixture was used for both dishes. Nonpolar lipids from cells in confluent 60-mm dishes were extracted with hexane/isopropyl alcohol 3:2 (Underwood et al. 1998). In Fig. 6 A and 7 A, extracted lipids (100% of the FRT and HEK 293 extracts or 10% of the COS extracts) were separated by high-performance thin-layer chromatography (HP-TLC) using hexane/isopropyl ether/acetic acid 65:35:2, and visualized by charring and quantitated by densitometric scanning and comparison to standards on the same plate as described (Brown and Rose 1992). Cholesterol, not expected to accumulate in lipid droplets, was also scanned as an internal control.

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