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Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell.

Fujimoto T, Kogo H, Ishiguro K, Tauchi K, Nomura R - J. Cell Biol. (2001)

Bottom Line: The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein.In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain.It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. tfujimot@med.nagoya-u.ac.jp

ABSTRACT
Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

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Confocal microscopy of HepG2 stably expressing caveolin-2β (a) and caveolin-1 (b). Caveolins (green), LD stained by Sudan III (red). Caveolin-2 is seen around LD discontinuously, whereas caveolin-1 is along the cell edge, and not related to LD. (c) FRTL-5 transiently transfected with full-length caveolin-2 cDNA and cultured with OA/BSA for 2 d. Caveolin-2 (green) is observed around LD (red). HepG2 stably transfected with caveolin-2β (d) or full-length caveolin-2 (e) cDNA was labeled for caveolin-2 (green) and GM130 (red). The labeling of caveolin-2β overlapping with that of GM130 is scarce (d), whereas caveolin-2α, the predominant isoform in the cell, is mostly localized in the Golgi (e). Arrows indicate labeling around LD. Bars, 10 μm.
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Figure 1: Confocal microscopy of HepG2 stably expressing caveolin-2β (a) and caveolin-1 (b). Caveolins (green), LD stained by Sudan III (red). Caveolin-2 is seen around LD discontinuously, whereas caveolin-1 is along the cell edge, and not related to LD. (c) FRTL-5 transiently transfected with full-length caveolin-2 cDNA and cultured with OA/BSA for 2 d. Caveolin-2 (green) is observed around LD (red). HepG2 stably transfected with caveolin-2β (d) or full-length caveolin-2 (e) cDNA was labeled for caveolin-2 (green) and GM130 (red). The labeling of caveolin-2β overlapping with that of GM130 is scarce (d), whereas caveolin-2α, the predominant isoform in the cell, is mostly localized in the Golgi (e). Arrows indicate labeling around LD. Bars, 10 μm.

Mentions: HepG2 lacks caveolins and has prominent LD in a standard culture condition. Human caveolin-2β transfected to HepG2 showed round-shaped labeling, which was revealed to be around LD by Sudan III staining (Fig. 1 a). Labeling for caveolin-2 was seen discontinuously around LD. Four cell lines stably expressing caveolin-2β gave equivalent results, and clone A-8 was used for most experiments. Expression of caveolin-2 did not appear to change the size and number of LD in HepG2. In contrast, human caveolin-1 transfected to HepG2 was seen in the cell surface and not related to LD (Fig. 1 b). Transfected caveolin-2 also occurred in LD of other cell types. In FRTL-5, LD were absent in a normal culture condition, and transfected caveolin-2 was seen in the Golgi as reported (not shown; Mora et al. 1999). But when LD were induced by OA/BSA, caveolin-2 was observed around them (Fig. 1 c).


Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell.

Fujimoto T, Kogo H, Ishiguro K, Tauchi K, Nomura R - J. Cell Biol. (2001)

Confocal microscopy of HepG2 stably expressing caveolin-2β (a) and caveolin-1 (b). Caveolins (green), LD stained by Sudan III (red). Caveolin-2 is seen around LD discontinuously, whereas caveolin-1 is along the cell edge, and not related to LD. (c) FRTL-5 transiently transfected with full-length caveolin-2 cDNA and cultured with OA/BSA for 2 d. Caveolin-2 (green) is observed around LD (red). HepG2 stably transfected with caveolin-2β (d) or full-length caveolin-2 (e) cDNA was labeled for caveolin-2 (green) and GM130 (red). The labeling of caveolin-2β overlapping with that of GM130 is scarce (d), whereas caveolin-2α, the predominant isoform in the cell, is mostly localized in the Golgi (e). Arrows indicate labeling around LD. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 1: Confocal microscopy of HepG2 stably expressing caveolin-2β (a) and caveolin-1 (b). Caveolins (green), LD stained by Sudan III (red). Caveolin-2 is seen around LD discontinuously, whereas caveolin-1 is along the cell edge, and not related to LD. (c) FRTL-5 transiently transfected with full-length caveolin-2 cDNA and cultured with OA/BSA for 2 d. Caveolin-2 (green) is observed around LD (red). HepG2 stably transfected with caveolin-2β (d) or full-length caveolin-2 (e) cDNA was labeled for caveolin-2 (green) and GM130 (red). The labeling of caveolin-2β overlapping with that of GM130 is scarce (d), whereas caveolin-2α, the predominant isoform in the cell, is mostly localized in the Golgi (e). Arrows indicate labeling around LD. Bars, 10 μm.
Mentions: HepG2 lacks caveolins and has prominent LD in a standard culture condition. Human caveolin-2β transfected to HepG2 showed round-shaped labeling, which was revealed to be around LD by Sudan III staining (Fig. 1 a). Labeling for caveolin-2 was seen discontinuously around LD. Four cell lines stably expressing caveolin-2β gave equivalent results, and clone A-8 was used for most experiments. Expression of caveolin-2 did not appear to change the size and number of LD in HepG2. In contrast, human caveolin-1 transfected to HepG2 was seen in the cell surface and not related to LD (Fig. 1 b). Transfected caveolin-2 also occurred in LD of other cell types. In FRTL-5, LD were absent in a normal culture condition, and transfected caveolin-2 was seen in the Golgi as reported (not shown; Mora et al. 1999). But when LD were induced by OA/BSA, caveolin-2 was observed around them (Fig. 1 c).

Bottom Line: The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein.In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain.It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. tfujimot@med.nagoya-u.ac.jp

ABSTRACT
Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

Show MeSH