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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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KKSL-tagged caveolin-1 mutants in isolated LDs. LDs were isolated from oleic acid–treated FRT cells expressing Cav-KKSL, 97/SASA-KKSL, Ins-7L(1+2)-KKSL, or Ins-7L(1+2)+Δ112-125-KKSL as indicated. Proteins in the LDs (LD) or in a reserved 5% of the whole cell homogenate (WC) were analyzed by SDS-PAGE and Western blotting and detected by ECL. Blots were cut in three parts. Top, probed with anticalnexin and HRP-donkey anti–rabbit IgG. Middle, anti-ADRP and HRP-GAM. Bottom, anticaveolin and HRP-donkey anti–rabbit IgG. Schematic depictions of proteins are as in Fig. 7.
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fig8: KKSL-tagged caveolin-1 mutants in isolated LDs. LDs were isolated from oleic acid–treated FRT cells expressing Cav-KKSL, 97/SASA-KKSL, Ins-7L(1+2)-KKSL, or Ins-7L(1+2)+Δ112-125-KKSL as indicated. Proteins in the LDs (LD) or in a reserved 5% of the whole cell homogenate (WC) were analyzed by SDS-PAGE and Western blotting and detected by ECL. Blots were cut in three parts. Top, probed with anticalnexin and HRP-donkey anti–rabbit IgG. Middle, anti-ADRP and HRP-GAM. Bottom, anticaveolin and HRP-donkey anti–rabbit IgG. Schematic depictions of proteins are as in Fig. 7.

Mentions: To confirm the IF results, we examined LDs isolated from transfected FRT cells for the presence of wild-type Cav-KKSL or selected KKSL-tagged mutants (Fig. 8). Aliquots of whole cell homogenate (Fig. 8, WC) and isolated LDs (Fig. 8, LD) from transfected FRT cells were probed for calnexin (to monitor ER contamination of the LD fraction), ADRP (to monitor LD yield), and various caveolin mutants (to monitor expression and LD targeting). In agreement with the IF results, wild-type Cav-KKSL and 97/SASA-KKSL were present in LDs, whereas Ins-7L(1+2)-KKSL and Ins-7L(1+2)+Δ112-125-KKSL were largely excluded from LDs (Fig. 8).


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)

KKSL-tagged caveolin-1 mutants in isolated LDs. LDs were isolated from oleic acid–treated FRT cells expressing Cav-KKSL, 97/SASA-KKSL, Ins-7L(1+2)-KKSL, or Ins-7L(1+2)+Δ112-125-KKSL as indicated. Proteins in the LDs (LD) or in a reserved 5% of the whole cell homogenate (WC) were analyzed by SDS-PAGE and Western blotting and detected by ECL. Blots were cut in three parts. Top, probed with anticalnexin and HRP-donkey anti–rabbit IgG. Middle, anti-ADRP and HRP-GAM. Bottom, anticaveolin and HRP-donkey anti–rabbit IgG. Schematic depictions of proteins are as in Fig. 7.
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Related In: Results  -  Collection

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fig8: KKSL-tagged caveolin-1 mutants in isolated LDs. LDs were isolated from oleic acid–treated FRT cells expressing Cav-KKSL, 97/SASA-KKSL, Ins-7L(1+2)-KKSL, or Ins-7L(1+2)+Δ112-125-KKSL as indicated. Proteins in the LDs (LD) or in a reserved 5% of the whole cell homogenate (WC) were analyzed by SDS-PAGE and Western blotting and detected by ECL. Blots were cut in three parts. Top, probed with anticalnexin and HRP-donkey anti–rabbit IgG. Middle, anti-ADRP and HRP-GAM. Bottom, anticaveolin and HRP-donkey anti–rabbit IgG. Schematic depictions of proteins are as in Fig. 7.
Mentions: To confirm the IF results, we examined LDs isolated from transfected FRT cells for the presence of wild-type Cav-KKSL or selected KKSL-tagged mutants (Fig. 8). Aliquots of whole cell homogenate (Fig. 8, WC) and isolated LDs (Fig. 8, LD) from transfected FRT cells were probed for calnexin (to monitor ER contamination of the LD fraction), ADRP (to monitor LD yield), and various caveolin mutants (to monitor expression and LD targeting). In agreement with the IF results, wild-type Cav-KKSL and 97/SASA-KKSL were present in LDs, whereas Ins-7L(1+2)-KKSL and Ins-7L(1+2)+Δ112-125-KKSL were largely excluded from LDs (Fig. 8).

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