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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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How caveolin-1 might enter lipid droplets. Neutral lipids may accumulate in the interior of the ER bilayer, making a bulge that eventually buds into the cytoplasm to form a droplet surrounded by an ER-derived phospholipid monolayer. This process would drive opposite ER membrane monolayers apart, effectively thickening the hydrophobic portion of the membrane. Most membrane proteins, which contain hydrophilic domains on both sides of the bilayer, could not be accommodated in this environment and would be excluded from the forming droplets. By contrast, caveolin-1 has no luminal hydrophilic domain and could easily diffuse laterally between the ER membrane proper and the monolayer surrounding the nascent droplets.
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Figure 8: How caveolin-1 might enter lipid droplets. Neutral lipids may accumulate in the interior of the ER bilayer, making a bulge that eventually buds into the cytoplasm to form a droplet surrounded by an ER-derived phospholipid monolayer. This process would drive opposite ER membrane monolayers apart, effectively thickening the hydrophobic portion of the membrane. Most membrane proteins, which contain hydrophilic domains on both sides of the bilayer, could not be accommodated in this environment and would be excluded from the forming droplets. By contrast, caveolin-1 has no luminal hydrophilic domain and could easily diffuse laterally between the ER membrane proper and the monolayer surrounding the nascent droplets.

Mentions: Lipid droplet biogenesis is not well understood (Londos et al. 1999; Murphy and Vance 1999). However, the prevailing model rationalizes how caveolins (but not most other membrane proteins) might be able to enter the droplets (Fig. 8). Lipid droplets are derived from the ER, where TG and CE are synthesized, and are thought to be surrounded by a phospholipid monolayer (Londos et al. 1999; Murphy and Vance 1999). These observations suggest that neutral lipids accumulate in the hydrophobic core of the bilayer, forming a bulge that eventually buds from the ER membrane to form a free droplet. Accumulation of neutral lipids in the bilayer core would initially force opposite leaflets of the bilayer apart, increasing bilayer thickness. Such a thickened bilayer would not be able to accommodate transmembrane proteins that have hydrophilic domains on both sides of the membrane, and these proteins would be excluded from the forming droplets. Caveolins, by contrast, lack luminal hydrophilic domains, and could diffuse freely between the ER membrane and the monolayer surrounding the nascent droplet. Thus, caveolins would not need to dissociate from the ER membrane and expose their hydrophobic domains to the cytosol to enter the droplets. This model suggests that caveolins can enter lipid droplets only while they are forming, and are still in contact with the ER. Consistent with this idea, we noticed that in some transfected cells, only some of the Nile red positive droplets contained Cav–KKSL (Fig. 2). We speculate that the Cav–KKSL-negative droplets budded from the ER before expression of Cav–KKSL started.


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)

How caveolin-1 might enter lipid droplets. Neutral lipids may accumulate in the interior of the ER bilayer, making a bulge that eventually buds into the cytoplasm to form a droplet surrounded by an ER-derived phospholipid monolayer. This process would drive opposite ER membrane monolayers apart, effectively thickening the hydrophobic portion of the membrane. Most membrane proteins, which contain hydrophilic domains on both sides of the bilayer, could not be accommodated in this environment and would be excluded from the forming droplets. By contrast, caveolin-1 has no luminal hydrophilic domain and could easily diffuse laterally between the ER membrane proper and the monolayer surrounding the nascent droplets.
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Related In: Results  -  Collection

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Figure 8: How caveolin-1 might enter lipid droplets. Neutral lipids may accumulate in the interior of the ER bilayer, making a bulge that eventually buds into the cytoplasm to form a droplet surrounded by an ER-derived phospholipid monolayer. This process would drive opposite ER membrane monolayers apart, effectively thickening the hydrophobic portion of the membrane. Most membrane proteins, which contain hydrophilic domains on both sides of the bilayer, could not be accommodated in this environment and would be excluded from the forming droplets. By contrast, caveolin-1 has no luminal hydrophilic domain and could easily diffuse laterally between the ER membrane proper and the monolayer surrounding the nascent droplets.
Mentions: Lipid droplet biogenesis is not well understood (Londos et al. 1999; Murphy and Vance 1999). However, the prevailing model rationalizes how caveolins (but not most other membrane proteins) might be able to enter the droplets (Fig. 8). Lipid droplets are derived from the ER, where TG and CE are synthesized, and are thought to be surrounded by a phospholipid monolayer (Londos et al. 1999; Murphy and Vance 1999). These observations suggest that neutral lipids accumulate in the hydrophobic core of the bilayer, forming a bulge that eventually buds from the ER membrane to form a free droplet. Accumulation of neutral lipids in the bilayer core would initially force opposite leaflets of the bilayer apart, increasing bilayer thickness. Such a thickened bilayer would not be able to accommodate transmembrane proteins that have hydrophilic domains on both sides of the membrane, and these proteins would be excluded from the forming droplets. Caveolins, by contrast, lack luminal hydrophilic domains, and could diffuse freely between the ER membrane and the monolayer surrounding the nascent droplet. Thus, caveolins would not need to dissociate from the ER membrane and expose their hydrophobic domains to the cytosol to enter the droplets. This model suggests that caveolins can enter lipid droplets only while they are forming, and are still in contact with the ER. Consistent with this idea, we noticed that in some transfected cells, only some of the Nile red positive droplets contained Cav–KKSL (Fig. 2). We speculate that the Cav–KKSL-negative droplets budded from the ER before expression of Cav–KKSL started.

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