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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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Localization of wild-type and mutant caveolin-1 in BFA-treated FRT cells. (A) Caveolin-1; (B) Δ101-134; (C) 118A5; (D) 123A6; (E) Δ112-125; (F) Δ59; (G) Ins7L(1+2); (H) Ins-7L1; (I) Ins-7L2; (J) Ins-14L; (K) Ins7L(1+2)+ Δ112-125; and (L) ΔC. Cells were treated with BFA for 5 h. Proteins were visualized by IF, using anticaveolin antibodies and DTAF-GAR. Arrows, LDs. Arrowheads, puncta staining for GM130 (not depicted), presumed to be ER exit sites.
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fig4: Localization of wild-type and mutant caveolin-1 in BFA-treated FRT cells. (A) Caveolin-1; (B) Δ101-134; (C) 118A5; (D) 123A6; (E) Δ112-125; (F) Δ59; (G) Ins7L(1+2); (H) Ins-7L1; (I) Ins-7L2; (J) Ins-14L; (K) Ins7L(1+2)+ Δ112-125; and (L) ΔC. Cells were treated with BFA for 5 h. Proteins were visualized by IF, using anticaveolin antibodies and DTAF-GAR. Arrows, LDs. Arrowheads, puncta staining for GM130 (not depicted), presumed to be ER exit sites.

Mentions: In the rest of this work, we examined caveolin-1 mutants, attempting to identify sequences needed for LD targeting. We first examined mutants in BFA-treated FRT cells, searching for those that failed to accumulate in LDs. Mutants are schematically diagrammed and the results are listed in Fig. 3, with pictures of selected mutants shown in Figs. 4 and 5. After BFA treatment, in FRT cells, as in COS cells, wild-type caveolin-1 was present in structures with the characteristic round shape of LDs (Fig. 4 A, arrows) that stained for the LD marker protein ADRP (Fig. 5, A–C) in 66 ± 15% (n = 6) of transfected cells after BFA treatment. Although the methanol fixation required for efficient detection of ADRP distorted LD shape, as reported previously (DiDonato and Brasaemle, 2003), colocalization of ADRP and caveolin was clear (Fig. 5, A–C). Caveolin-1 staining was also seen in caveolae, which did not stain for ADRP. Without BFA treatment, wild-type caveolin-1 was occasionally (∼5% of cells) seen in LDs in FRT cells, in the very highest expressing cells (unpublished data). After BFA treatment, caveolin-1 and all the mutants examined were also seen in punctate structures larger than caveolae and distributed throughout the cell (Fig. 4 A, arrowheads). These structures did not stain for ADRP and were thus not related to LDs, but stained for GM130 (unpublished data), and are probably ER exit sites (Ward et al., 2001). The unusual behavior of caveolin-1 in concentrating in these structures in BFA-treated cells will be described elsewhere (unpublished data).


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)

Localization of wild-type and mutant caveolin-1 in BFA-treated FRT cells. (A) Caveolin-1; (B) Δ101-134; (C) 118A5; (D) 123A6; (E) Δ112-125; (F) Δ59; (G) Ins7L(1+2); (H) Ins-7L1; (I) Ins-7L2; (J) Ins-14L; (K) Ins7L(1+2)+ Δ112-125; and (L) ΔC. Cells were treated with BFA for 5 h. Proteins were visualized by IF, using anticaveolin antibodies and DTAF-GAR. Arrows, LDs. Arrowheads, puncta staining for GM130 (not depicted), presumed to be ER exit sites.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171963&req=5

fig4: Localization of wild-type and mutant caveolin-1 in BFA-treated FRT cells. (A) Caveolin-1; (B) Δ101-134; (C) 118A5; (D) 123A6; (E) Δ112-125; (F) Δ59; (G) Ins7L(1+2); (H) Ins-7L1; (I) Ins-7L2; (J) Ins-14L; (K) Ins7L(1+2)+ Δ112-125; and (L) ΔC. Cells were treated with BFA for 5 h. Proteins were visualized by IF, using anticaveolin antibodies and DTAF-GAR. Arrows, LDs. Arrowheads, puncta staining for GM130 (not depicted), presumed to be ER exit sites.
Mentions: In the rest of this work, we examined caveolin-1 mutants, attempting to identify sequences needed for LD targeting. We first examined mutants in BFA-treated FRT cells, searching for those that failed to accumulate in LDs. Mutants are schematically diagrammed and the results are listed in Fig. 3, with pictures of selected mutants shown in Figs. 4 and 5. After BFA treatment, in FRT cells, as in COS cells, wild-type caveolin-1 was present in structures with the characteristic round shape of LDs (Fig. 4 A, arrows) that stained for the LD marker protein ADRP (Fig. 5, A–C) in 66 ± 15% (n = 6) of transfected cells after BFA treatment. Although the methanol fixation required for efficient detection of ADRP distorted LD shape, as reported previously (DiDonato and Brasaemle, 2003), colocalization of ADRP and caveolin was clear (Fig. 5, A–C). Caveolin-1 staining was also seen in caveolae, which did not stain for ADRP. Without BFA treatment, wild-type caveolin-1 was occasionally (∼5% of cells) seen in LDs in FRT cells, in the very highest expressing cells (unpublished data). After BFA treatment, caveolin-1 and all the mutants examined were also seen in punctate structures larger than caveolae and distributed throughout the cell (Fig. 4 A, arrowheads). These structures did not stain for ADRP and were thus not related to LDs, but stained for GM130 (unpublished data), and are probably ER exit sites (Ward et al., 2001). The unusual behavior of caveolin-1 in concentrating in these structures in BFA-treated cells will be described elsewhere (unpublished data).

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