Limits...
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.

Show MeSH
(a) A diagram of caveolin-2 mutants and their distribution on transient expression in HepG2. (b–e) Distribution of mutants was observed by anti–caveolin-2 labeling (b, d, and e) or by EGFP (c) (green), and compared with LD, Golgi, or ER markers (red). Arrows mark the labeling apparently encircling LD. Bars, 10 μm. (b) Single amino acid replacements to examine the difference between α and β isoforms. Even when methionine-14, the second translation initiation site, was replaced with leucine (M14/L), or glycine-2, a putative myristoylation site, was replaced with alanine (G2/A), distribution was not different from that of caveolin-2α. (c) NH2-terminal deletion mutants fused to the COOH terminus of EGFP. Deletion of up to 69 residues (70–162) did not affect the LD localization, but further truncation (71–162) caused cytosolic distribution. (d) COOH-terminal deletion mutants. For both α and β isoforms, the distribution was not changed when the sequence up to the 150th residue was maintained. As more amino acids were deleted, localization in LD and the Golgi became less distinct, and a network-like labeling increased. The latter labeling was similar to that of calreticulin (result of 14–127 is shown). When ER was made to retract from the cell edge (arrowheads) by nocodazole, the labeling for the mutant showed a matching redistribution. (e) Caveolin-2β lacking the central hydrophobic domain [beta-TM(−)] was localized in the Golgi, labeled by anti–GM130. Deletion of the same domain from caveolin-2α gave the same result.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198803&req=5

Figure 5: (a) A diagram of caveolin-2 mutants and their distribution on transient expression in HepG2. (b–e) Distribution of mutants was observed by anti–caveolin-2 labeling (b, d, and e) or by EGFP (c) (green), and compared with LD, Golgi, or ER markers (red). Arrows mark the labeling apparently encircling LD. Bars, 10 μm. (b) Single amino acid replacements to examine the difference between α and β isoforms. Even when methionine-14, the second translation initiation site, was replaced with leucine (M14/L), or glycine-2, a putative myristoylation site, was replaced with alanine (G2/A), distribution was not different from that of caveolin-2α. (c) NH2-terminal deletion mutants fused to the COOH terminus of EGFP. Deletion of up to 69 residues (70–162) did not affect the LD localization, but further truncation (71–162) caused cytosolic distribution. (d) COOH-terminal deletion mutants. For both α and β isoforms, the distribution was not changed when the sequence up to the 150th residue was maintained. As more amino acids were deleted, localization in LD and the Golgi became less distinct, and a network-like labeling increased. The latter labeling was similar to that of calreticulin (result of 14–127 is shown). When ER was made to retract from the cell edge (arrowheads) by nocodazole, the labeling for the mutant showed a matching redistribution. (e) Caveolin-2β lacking the central hydrophobic domain [beta-TM(−)] was localized in the Golgi, labeled by anti–GM130. Deletion of the same domain from caveolin-2α gave the same result.

Mentions: To identify molecular domains required for targeting to LD, mouse caveolin-2 mutants were constructed (Fig. 5 a) and transfected transiently to HepG2. First, to examine whether caveolin-2α goes to LD, the second methionine (residue 14) was replaced with leucine to abolish generation of the β isoform. The mutant (M14/L) showed the dual distribution in the Golgi and LD, proving that the α isoform can be distributed in LD (Fig. 5 b). Another mutant with glycine (residue 2) replaced with alanine (G2/A) was made to examine the possible influence of NH2-terminal myristoylation on targeting. G2/A showed a distribution similar to the intact α isoform and thus the myristoylation does not account for the preferential Golgi localization (Fig. 5 b). The result suggests that the 13 amino acids specific to the α isoform may reduce the affinity to LD, or may be a strong signal for Golgi localization.


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) A diagram of caveolin-2 mutants and their distribution on transient expression in HepG2. (b–e) Distribution of mutants was observed by anti–caveolin-2 labeling (b, d, and e) or by EGFP (c) (green), and compared with LD, Golgi, or ER markers (red). Arrows mark the labeling apparently encircling LD. Bars, 10 μm. (b) Single amino acid replacements to examine the difference between α and β isoforms. Even when methionine-14, the second translation initiation site, was replaced with leucine (M14/L), or glycine-2, a putative myristoylation site, was replaced with alanine (G2/A), distribution was not different from that of caveolin-2α. (c) NH2-terminal deletion mutants fused to the COOH terminus of EGFP. Deletion of up to 69 residues (70–162) did not affect the LD localization, but further truncation (71–162) caused cytosolic distribution. (d) COOH-terminal deletion mutants. For both α and β isoforms, the distribution was not changed when the sequence up to the 150th residue was maintained. As more amino acids were deleted, localization in LD and the Golgi became less distinct, and a network-like labeling increased. The latter labeling was similar to that of calreticulin (result of 14–127 is shown). When ER was made to retract from the cell edge (arrowheads) by nocodazole, the labeling for the mutant showed a matching redistribution. (e) Caveolin-2β lacking the central hydrophobic domain [beta-TM(−)] was localized in the Golgi, labeled by anti–GM130. Deletion of the same domain from caveolin-2α gave the same result.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198803&req=5

Figure 5: (a) A diagram of caveolin-2 mutants and their distribution on transient expression in HepG2. (b–e) Distribution of mutants was observed by anti–caveolin-2 labeling (b, d, and e) or by EGFP (c) (green), and compared with LD, Golgi, or ER markers (red). Arrows mark the labeling apparently encircling LD. Bars, 10 μm. (b) Single amino acid replacements to examine the difference between α and β isoforms. Even when methionine-14, the second translation initiation site, was replaced with leucine (M14/L), or glycine-2, a putative myristoylation site, was replaced with alanine (G2/A), distribution was not different from that of caveolin-2α. (c) NH2-terminal deletion mutants fused to the COOH terminus of EGFP. Deletion of up to 69 residues (70–162) did not affect the LD localization, but further truncation (71–162) caused cytosolic distribution. (d) COOH-terminal deletion mutants. For both α and β isoforms, the distribution was not changed when the sequence up to the 150th residue was maintained. As more amino acids were deleted, localization in LD and the Golgi became less distinct, and a network-like labeling increased. The latter labeling was similar to that of calreticulin (result of 14–127 is shown). When ER was made to retract from the cell edge (arrowheads) by nocodazole, the labeling for the mutant showed a matching redistribution. (e) Caveolin-2β lacking the central hydrophobic domain [beta-TM(−)] was localized in the Golgi, labeled by anti–GM130. Deletion of the same domain from caveolin-2α gave the same result.
Mentions: To identify molecular domains required for targeting to LD, mouse caveolin-2 mutants were constructed (Fig. 5 a) and transfected transiently to HepG2. First, to examine whether caveolin-2α goes to LD, the second methionine (residue 14) was replaced with leucine to abolish generation of the β isoform. The mutant (M14/L) showed the dual distribution in the Golgi and LD, proving that the α isoform can be distributed in LD (Fig. 5 b). Another mutant with glycine (residue 2) replaced with alanine (G2/A) was made to examine the possible influence of NH2-terminal myristoylation on targeting. G2/A showed a distribution similar to the intact α isoform and thus the myristoylation does not account for the preferential Golgi localization (Fig. 5 b). The result suggests that the 13 amino acids specific to the α isoform may reduce the affinity to LD, or may be a strong signal for Golgi localization.

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