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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 KKSL-tagged caveolin-1 mutants. FRT cells expressing Ins-7L1-KKSL (A–C), 97/SASA-KKSL (D–F), Ins-7L(1+2)-KKSL (G–I), or Ins-7L(1+2)+Δ112-125-KKSL (J–L) were methanol fixed. Caveolin-1 proteins (A, D, G, and J) and ADRP in the same cells (B, E, H, and K) were detected by IF. (C, F, I, and L) Merged images. Schematic depictions of proteins are as in Fig. 3, except that only two triangles indicate the four substitutions in 97/SASA. *, KKSL tag.
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fig7: Localization of KKSL-tagged caveolin-1 mutants. FRT cells expressing Ins-7L1-KKSL (A–C), 97/SASA-KKSL (D–F), Ins-7L(1+2)-KKSL (G–I), or Ins-7L(1+2)+Δ112-125-KKSL (J–L) were methanol fixed. Caveolin-1 proteins (A, D, G, and J) and ADRP in the same cells (B, E, H, and K) were detected by IF. (C, F, I, and L) Merged images. Schematic depictions of proteins are as in Fig. 3, except that only two triangles indicate the four substitutions in 97/SASA. *, KKSL tag.

Mentions: To ensure that our results did not depend on BFA treatment, we appended KKSL ER retrieval tags to selected caveolin-1 mutants. Wild-type Cav-KKSL accumulates in LDs even without BFA (Ostermeyer et al., 2001). Similarly, KKSL-tagged forms of 97/SASA and Ins-7L1 were detected in LDs in virtually every expressing cell (Fig. 7). This contrasted with the localization of the non-KKSL tagged versions of the same proteins in BFA-treated cells, in which LD localization was detected in 60–80% of the cells. Increased LD accumulation of the KKSL-tagged proteins probably resulted from the fact that they were continuously retrieved to the ER, instead of accumulating there for only 5 h during BFA treatment. Nevertheless, KKSL-tagged forms of Ins-7L(1+2) and Ins-7L(1+2)+Δ112-125 were never seen in LDs (Fig. 7), although Ins-7L(1+2)-KKSL was expressed about as well as wild-type Cav-KKSL, 97/SASA-KKSL, and Ins-7L1-KKSL. Ins-7L(1+2)+Δ112-125-KKSL was expressed at a higher level (unpublished data). Thus, the inability of certain mutants to accumulate in LDs did not depend on BFA treatment.


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 KKSL-tagged caveolin-1 mutants. FRT cells expressing Ins-7L1-KKSL (A–C), 97/SASA-KKSL (D–F), Ins-7L(1+2)-KKSL (G–I), or Ins-7L(1+2)+Δ112-125-KKSL (J–L) were methanol fixed. Caveolin-1 proteins (A, D, G, and J) and ADRP in the same cells (B, E, H, and K) were detected by IF. (C, F, I, and L) Merged images. Schematic depictions of proteins are as in Fig. 3, except that only two triangles indicate the four substitutions in 97/SASA. *, KKSL tag.
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fig7: Localization of KKSL-tagged caveolin-1 mutants. FRT cells expressing Ins-7L1-KKSL (A–C), 97/SASA-KKSL (D–F), Ins-7L(1+2)-KKSL (G–I), or Ins-7L(1+2)+Δ112-125-KKSL (J–L) were methanol fixed. Caveolin-1 proteins (A, D, G, and J) and ADRP in the same cells (B, E, H, and K) were detected by IF. (C, F, I, and L) Merged images. Schematic depictions of proteins are as in Fig. 3, except that only two triangles indicate the four substitutions in 97/SASA. *, KKSL tag.
Mentions: To ensure that our results did not depend on BFA treatment, we appended KKSL ER retrieval tags to selected caveolin-1 mutants. Wild-type Cav-KKSL accumulates in LDs even without BFA (Ostermeyer et al., 2001). Similarly, KKSL-tagged forms of 97/SASA and Ins-7L1 were detected in LDs in virtually every expressing cell (Fig. 7). This contrasted with the localization of the non-KKSL tagged versions of the same proteins in BFA-treated cells, in which LD localization was detected in 60–80% of the cells. Increased LD accumulation of the KKSL-tagged proteins probably resulted from the fact that they were continuously retrieved to the ER, instead of accumulating there for only 5 h during BFA treatment. Nevertheless, KKSL-tagged forms of Ins-7L(1+2) and Ins-7L(1+2)+Δ112-125 were never seen in LDs (Fig. 7), although Ins-7L(1+2)-KKSL was expressed about as well as wild-type Cav-KKSL, 97/SASA-KKSL, and Ins-7L1-KKSL. Ins-7L(1+2)+Δ112-125-KKSL was expressed at a higher level (unpublished data). Thus, the inability of certain mutants to accumulate in LDs did not depend on BFA treatment.

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