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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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(a and b) Immunoelectron microscopy of HepG2 expressing caveolin-2β (clone A-8). Bar, 500 nm. (a) Ultrathin cryosection. Gold particles labeling caveolin-2 are observed in the rim of LD in small clusters (arrowheads); the content of LD appears vacant. (b) Freeze-fracture replica. Gold particles for caveolin-2 are observed in clusters (arrowheads) on the P face of LD, which show an onion-like morphology. The E face of LD (E) is devoid of labeling. (c) Western blotting of subcellular fractions obtained from HepG2 clone A-8. The graph shows the protein content of the fractions. The top two fractions (#1 and #2) contained little protein, but showed positive signals for caveolin-2 and adipophilin. GM130, 66-kD protein, and Na+/K+-ATPase (Golgi, ER, and plasma membrane markers, respectively) are found only in the bottom fractions.
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Figure 2: (a and b) Immunoelectron microscopy of HepG2 expressing caveolin-2β (clone A-8). Bar, 500 nm. (a) Ultrathin cryosection. Gold particles labeling caveolin-2 are observed in the rim of LD in small clusters (arrowheads); the content of LD appears vacant. (b) Freeze-fracture replica. Gold particles for caveolin-2 are observed in clusters (arrowheads) on the P face of LD, which show an onion-like morphology. The E face of LD (E) is devoid of labeling. (c) Western blotting of subcellular fractions obtained from HepG2 clone A-8. The graph shows the protein content of the fractions. The top two fractions (#1 and #2) contained little protein, but showed positive signals for caveolin-2 and adipophilin. GM130, 66-kD protein, and Na+/K+-ATPase (Golgi, ER, and plasma membrane markers, respectively) are found only in the bottom fractions.

Mentions: Localization of caveolin-2 in LD was confirmed by immunoelectron microscopy. In cryosections, the labeling was localized along the LD rim, and not in adjacent organelles (Fig. 2 a). Notably, most gold particles were seen in small clusters. The localization in LD was even more evident in freeze-fracture replicas. LD was identified by the onion-like morphology; the labeling for caveolin-2 also occurred in clusters and was seen only on the P, but not the E, face of LD (Fig. 2 b).


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)

(a and b) Immunoelectron microscopy of HepG2 expressing caveolin-2β (clone A-8). Bar, 500 nm. (a) Ultrathin cryosection. Gold particles labeling caveolin-2 are observed in the rim of LD in small clusters (arrowheads); the content of LD appears vacant. (b) Freeze-fracture replica. Gold particles for caveolin-2 are observed in clusters (arrowheads) on the P face of LD, which show an onion-like morphology. The E face of LD (E) is devoid of labeling. (c) Western blotting of subcellular fractions obtained from HepG2 clone A-8. The graph shows the protein content of the fractions. The top two fractions (#1 and #2) contained little protein, but showed positive signals for caveolin-2 and adipophilin. GM130, 66-kD protein, and Na+/K+-ATPase (Golgi, ER, and plasma membrane markers, respectively) are found only in the bottom fractions.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: (a and b) Immunoelectron microscopy of HepG2 expressing caveolin-2β (clone A-8). Bar, 500 nm. (a) Ultrathin cryosection. Gold particles labeling caveolin-2 are observed in the rim of LD in small clusters (arrowheads); the content of LD appears vacant. (b) Freeze-fracture replica. Gold particles for caveolin-2 are observed in clusters (arrowheads) on the P face of LD, which show an onion-like morphology. The E face of LD (E) is devoid of labeling. (c) Western blotting of subcellular fractions obtained from HepG2 clone A-8. The graph shows the protein content of the fractions. The top two fractions (#1 and #2) contained little protein, but showed positive signals for caveolin-2 and adipophilin. GM130, 66-kD protein, and Na+/K+-ATPase (Golgi, ER, and plasma membrane markers, respectively) are found only in the bottom fractions.
Mentions: Localization of caveolin-2 in LD was confirmed by immunoelectron microscopy. In cryosections, the labeling was localized along the LD rim, and not in adjacent organelles (Fig. 2 a). Notably, most gold particles were seen in small clusters. The localization in LD was even more evident in freeze-fracture replicas. LD was identified by the onion-like morphology; the labeling for caveolin-2 also occurred in clusters and was seen only on the P, but not the E, face of LD (Fig. 2 b).

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