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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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BFA-induced LD accumulation of wild-type and mutant caveolin-1. FRT cells expressing the indicated proteins were treated with BFA for 5 h, and caveolin-1 was detected by IF. Proteins are listed by name and diagrammed schematically (not to scale). The NH2-terminal, hydrophobic, and COOH-terminal domains of caveolin-1 are schematized as open, shaded, and open boxes, respectively. Deletions are schematized as gaps, substitutions as closed triangles, and 7-Leu insertions as open triangles. Except for the hydrophobic domain mutants (102A5–130A5), the number of triangles corresponds to the number of changes. Mutants in the hydrophobic domain (Hyd D), NH2-terminal domain (NTD), and COOH-terminal domain (CTD) are grouped together. Pic., IF images of these proteins are shown in the indicated panels of Fig. 4.
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fig3: BFA-induced LD accumulation of wild-type and mutant caveolin-1. FRT cells expressing the indicated proteins were treated with BFA for 5 h, and caveolin-1 was detected by IF. Proteins are listed by name and diagrammed schematically (not to scale). The NH2-terminal, hydrophobic, and COOH-terminal domains of caveolin-1 are schematized as open, shaded, and open boxes, respectively. Deletions are schematized as gaps, substitutions as closed triangles, and 7-Leu insertions as open triangles. Except for the hydrophobic domain mutants (102A5–130A5), the number of triangles corresponds to the number of changes. Mutants in the hydrophobic domain (Hyd D), NH2-terminal domain (NTD), and COOH-terminal domain (CTD) are grouped together. Pic., IF images of these proteins are shown in the indicated panels of Fig. 4.

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)

BFA-induced LD accumulation of wild-type and mutant caveolin-1. FRT cells expressing the indicated proteins were treated with BFA for 5 h, and caveolin-1 was detected by IF. Proteins are listed by name and diagrammed schematically (not to scale). The NH2-terminal, hydrophobic, and COOH-terminal domains of caveolin-1 are schematized as open, shaded, and open boxes, respectively. Deletions are schematized as gaps, substitutions as closed triangles, and 7-Leu insertions as open triangles. Except for the hydrophobic domain mutants (102A5–130A5), the number of triangles corresponds to the number of changes. Mutants in the hydrophobic domain (Hyd D), NH2-terminal domain (NTD), and COOH-terminal domain (CTD) are grouped together. Pic., IF images of these proteins are shown in the indicated panels of Fig. 4.
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Related In: Results  -  Collection

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

fig3: BFA-induced LD accumulation of wild-type and mutant caveolin-1. FRT cells expressing the indicated proteins were treated with BFA for 5 h, and caveolin-1 was detected by IF. Proteins are listed by name and diagrammed schematically (not to scale). The NH2-terminal, hydrophobic, and COOH-terminal domains of caveolin-1 are schematized as open, shaded, and open boxes, respectively. Deletions are schematized as gaps, substitutions as closed triangles, and 7-Leu insertions as open triangles. Except for the hydrophobic domain mutants (102A5–130A5), the number of triangles corresponds to the number of changes. Mutants in the hydrophobic domain (Hyd D), NH2-terminal domain (NTD), and COOH-terminal domain (CTD) are grouped together. Pic., IF images of these proteins are shown in the indicated panels of Fig. 4.
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