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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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Related in: MedlinePlus

Expression level of caveolin-1 mutants does not correlate with LD targeting. FRT cells were cotransfected with PLAP-HA (as a transfection and loading control) and either Ins-7L1 (lane 1), Ins-7L2 (lane 2), Ins-7L(1+2)+Δ112-125 (lane 3), Δ59 (lane 4), Ins-7L(1+2) (lane 5), or Δ112-125 (lane 6); or, in a separate experiment, analyzed on a separate gel with Ins-7L(1+2) (lane 7) or ΔC (lane 8). Cells were lysed with gel loading buffer and proteins in equal aliquots of lysate were analyzed by SDS-PAGE and Western blotting. Blots were cut, and the tops were probed with anti-PLAP antibodies and the bottoms with anticaveolin antibodies. Both halves were probed with HRP–donkey anti–rabbit IgG, and proteins were visualized by ECL.
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fig6: Expression level of caveolin-1 mutants does not correlate with LD targeting. FRT cells were cotransfected with PLAP-HA (as a transfection and loading control) and either Ins-7L1 (lane 1), Ins-7L2 (lane 2), Ins-7L(1+2)+Δ112-125 (lane 3), Δ59 (lane 4), Ins-7L(1+2) (lane 5), or Δ112-125 (lane 6); or, in a separate experiment, analyzed on a separate gel with Ins-7L(1+2) (lane 7) or ΔC (lane 8). Cells were lysed with gel loading buffer and proteins in equal aliquots of lysate were analyzed by SDS-PAGE and Western blotting. Blots were cut, and the tops were probed with anti-PLAP antibodies and the bottoms with anticaveolin antibodies. Both halves were probed with HRP–donkey anti–rabbit IgG, and proteins were visualized by ECL.

Mentions: To ensure that apparent exclusion of certain mutants from LDs did not result from low expression, we compared the expression levels of selected mutants (Fig. 6). Mutants were coexpressed with PLAP-HA as a transfection and loading control. Expression levels of different mutants varied widely. Although expression of Ins-7L(1+2) was quite low, two other mutants that failed to accumulate in LDs, Δ59 and Ins-7L(1+2)+Δ112-125, were expressed well (Fig. 6, lanes 1–6). Thus, expression levels of the hydrophobic domain mutants did not correlate with LD accumulation.


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)

Expression level of caveolin-1 mutants does not correlate with LD targeting. FRT cells were cotransfected with PLAP-HA (as a transfection and loading control) and either Ins-7L1 (lane 1), Ins-7L2 (lane 2), Ins-7L(1+2)+Δ112-125 (lane 3), Δ59 (lane 4), Ins-7L(1+2) (lane 5), or Δ112-125 (lane 6); or, in a separate experiment, analyzed on a separate gel with Ins-7L(1+2) (lane 7) or ΔC (lane 8). Cells were lysed with gel loading buffer and proteins in equal aliquots of lysate were analyzed by SDS-PAGE and Western blotting. Blots were cut, and the tops were probed with anti-PLAP antibodies and the bottoms with anticaveolin antibodies. Both halves were probed with HRP–donkey anti–rabbit IgG, and proteins were visualized by ECL.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Expression level of caveolin-1 mutants does not correlate with LD targeting. FRT cells were cotransfected with PLAP-HA (as a transfection and loading control) and either Ins-7L1 (lane 1), Ins-7L2 (lane 2), Ins-7L(1+2)+Δ112-125 (lane 3), Δ59 (lane 4), Ins-7L(1+2) (lane 5), or Δ112-125 (lane 6); or, in a separate experiment, analyzed on a separate gel with Ins-7L(1+2) (lane 7) or ΔC (lane 8). Cells were lysed with gel loading buffer and proteins in equal aliquots of lysate were analyzed by SDS-PAGE and Western blotting. Blots were cut, and the tops were probed with anti-PLAP antibodies and the bottoms with anticaveolin antibodies. Both halves were probed with HRP–donkey anti–rabbit IgG, and proteins were visualized by ECL.
Mentions: To ensure that apparent exclusion of certain mutants from LDs did not result from low expression, we compared the expression levels of selected mutants (Fig. 6). Mutants were coexpressed with PLAP-HA as a transfection and loading control. Expression levels of different mutants varied widely. Although expression of Ins-7L(1+2) was quite low, two other mutants that failed to accumulate in LDs, Δ59 and Ins-7L(1+2)+Δ112-125, were expressed well (Fig. 6, lanes 1–6). Thus, expression levels of the hydrophobic domain mutants did not correlate with LD accumulation.

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