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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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Neither H-Ras nor the transmembrane protein PLAP-HA accumulate in LDs. (A, B, D, and E) GFP-tagged nonpalmitoylated H-Ras (A and B) and Cav-KKSL (D and E) were coexpressed in COS cells. Cells were not treated (A and D; same cell) or treated (B and E; same cell) with BFA for 5 h. Mutant H-Ras was visualized by GFP fluorescence, and caveolin-KKSL by IF, using Texas red–GAR. (C and F) In one cotransfected, BFA-treated FRT cell, PLAP-HA was detected with anti-PLAP and FITC-GAR (C), and Cav-KKSL was detected with anti-myc and Texas red–GAM (F).
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fig2: Neither H-Ras nor the transmembrane protein PLAP-HA accumulate in LDs. (A, B, D, and E) GFP-tagged nonpalmitoylated H-Ras (A and B) and Cav-KKSL (D and E) were coexpressed in COS cells. Cells were not treated (A and D; same cell) or treated (B and E; same cell) with BFA for 5 h. Mutant H-Ras was visualized by GFP fluorescence, and caveolin-KKSL by IF, using Texas red–GAR. (C and F) In one cotransfected, BFA-treated FRT cell, PLAP-HA was detected with anti-PLAP and FITC-GAR (C), and Cav-KKSL was detected with anti-myc and Texas red–GAM (F).

Mentions: To see if any overexpressed protein on the cytoplasmic surface of the ER entered LDs nonspecifically, we examined H-Ras, which is recruited to the ER after synthesis and reaches the plasma membrane via the secretory pathway (Choy et al., 1999). Ras proteins move slowly through the secretory pathway and are seen in the ER and Golgi apparatus at steady state. We saw no LD staining of EGFP–H-Ras expressed in COS cells (unpublished data). Because plasma membrane staining might have obscured LD staining, we expressed an EGFP mutant nonpalmitoylated H-Ras, which remains in the ER and Golgi apparatus and does not reach the cell surface (Choy et al., 1999). To identify LDs, we coexpressed Cav-KKSL (caveolin-1 with an appended ER retrieval motif), which accumulates in LDs even without BFA (Ostermeyer et al., 2001). Nonpalmitoylated H-Ras had a diffuse ER localization (Fig. 2, A and B) and was detected only rarely (<5% of transfected cells) in Cav-KKSL–positive LDs with or without 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)

Neither H-Ras nor the transmembrane protein PLAP-HA accumulate in LDs. (A, B, D, and E) GFP-tagged nonpalmitoylated H-Ras (A and B) and Cav-KKSL (D and E) were coexpressed in COS cells. Cells were not treated (A and D; same cell) or treated (B and E; same cell) with BFA for 5 h. Mutant H-Ras was visualized by GFP fluorescence, and caveolin-KKSL by IF, using Texas red–GAR. (C and F) In one cotransfected, BFA-treated FRT cell, PLAP-HA was detected with anti-PLAP and FITC-GAR (C), and Cav-KKSL was detected with anti-myc and Texas red–GAM (F).
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Related In: Results  -  Collection

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

fig2: Neither H-Ras nor the transmembrane protein PLAP-HA accumulate in LDs. (A, B, D, and E) GFP-tagged nonpalmitoylated H-Ras (A and B) and Cav-KKSL (D and E) were coexpressed in COS cells. Cells were not treated (A and D; same cell) or treated (B and E; same cell) with BFA for 5 h. Mutant H-Ras was visualized by GFP fluorescence, and caveolin-KKSL by IF, using Texas red–GAR. (C and F) In one cotransfected, BFA-treated FRT cell, PLAP-HA was detected with anti-PLAP and FITC-GAR (C), and Cav-KKSL was detected with anti-myc and Texas red–GAM (F).
Mentions: To see if any overexpressed protein on the cytoplasmic surface of the ER entered LDs nonspecifically, we examined H-Ras, which is recruited to the ER after synthesis and reaches the plasma membrane via the secretory pathway (Choy et al., 1999). Ras proteins move slowly through the secretory pathway and are seen in the ER and Golgi apparatus at steady state. We saw no LD staining of EGFP–H-Ras expressed in COS cells (unpublished data). Because plasma membrane staining might have obscured LD staining, we expressed an EGFP mutant nonpalmitoylated H-Ras, which remains in the ER and Golgi apparatus and does not reach the cell surface (Choy et al., 1999). To identify LDs, we coexpressed Cav-KKSL (caveolin-1 with an appended ER retrieval motif), which accumulates in LDs even without BFA (Ostermeyer et al., 2001). Nonpalmitoylated H-Ras had a diffuse ER localization (Fig. 2, A and B) and was detected only rarely (<5% of transfected cells) in Cav-KKSL–positive LDs with or without 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