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Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets.

Ostermeyer AG, Ramcharan LT, Zeng Y, Lublin DM, Brown DA - J. Cell Biol. (2004)

Bottom Line: Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs.The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1.We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA.

ABSTRACT
Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

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Effect of drugs on LD accumulation of caveolin-1. COS cells were left untreated (A–C) or treated with BFA alone (D–F) or BFA with nocodazole (G–I), cytochalasin B (J–L), or cycloheximide (M–O) for 5 h. Caveolin-1 was detected by IF with anti–caveolin-1 and Alexa Fluor 350-GAR (left) and LDs with Nile red (center). Right, merged images. Images were taken with a 100× objective. Each image shows a portion of one cell; for orientation, dark areas at lower right in F and I are the edges of nuclei. Bar, 5 μm.
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fig1: Effect of drugs on LD accumulation of caveolin-1. COS cells were left untreated (A–C) or treated with BFA alone (D–F) or BFA with nocodazole (G–I), cytochalasin B (J–L), or cycloheximide (M–O) for 5 h. Caveolin-1 was detected by IF with anti–caveolin-1 and Alexa Fluor 350-GAR (left) and LDs with Nile red (center). Right, merged images. Images were taken with a 100× objective. Each image shows a portion of one cell; for orientation, dark areas at lower right in F and I are the edges of nuclei. Bar, 5 μm.

Mentions: Caveolin-1 is normally concentrated in caveolae, and not in LDs, in COS cells (Fig. 1, A–C), but accumulates in LDs after 5 h of BFA treatment (Ostermeyer et al., 2001; Fig. 1, D–F). LDs were identified with the lipophilic dye Nile red (Fig. 1, B and E). Although not clear in Fig. 1, caveolin-1 also remained abundant in caveolae after BFA treatment. Here, we used the ability of BFA to induce LD accumulation of caveolin-1 to identify factors that blocked that targeting.


Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets.

Ostermeyer AG, Ramcharan LT, Zeng Y, Lublin DM, Brown DA - J. Cell Biol. (2004)

Effect of drugs on LD accumulation of caveolin-1. COS cells were left untreated (A–C) or treated with BFA alone (D–F) or BFA with nocodazole (G–I), cytochalasin B (J–L), or cycloheximide (M–O) for 5 h. Caveolin-1 was detected by IF with anti–caveolin-1 and Alexa Fluor 350-GAR (left) and LDs with Nile red (center). Right, merged images. Images were taken with a 100× objective. Each image shows a portion of one cell; for orientation, dark areas at lower right in F and I are the edges of nuclei. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171963&req=5

fig1: Effect of drugs on LD accumulation of caveolin-1. COS cells were left untreated (A–C) or treated with BFA alone (D–F) or BFA with nocodazole (G–I), cytochalasin B (J–L), or cycloheximide (M–O) for 5 h. Caveolin-1 was detected by IF with anti–caveolin-1 and Alexa Fluor 350-GAR (left) and LDs with Nile red (center). Right, merged images. Images were taken with a 100× objective. Each image shows a portion of one cell; for orientation, dark areas at lower right in F and I are the edges of nuclei. Bar, 5 μm.
Mentions: Caveolin-1 is normally concentrated in caveolae, and not in LDs, in COS cells (Fig. 1, A–C), but accumulates in LDs after 5 h of BFA treatment (Ostermeyer et al., 2001; Fig. 1, D–F). LDs were identified with the lipophilic dye Nile red (Fig. 1, B and E). Although not clear in Fig. 1, caveolin-1 also remained abundant in caveolae after BFA treatment. Here, we used the ability of BFA to induce LD accumulation of caveolin-1 to identify factors that blocked that targeting.

Bottom Line: Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs.The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1.We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA.

ABSTRACT
Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

Show MeSH
Related in: MedlinePlus