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Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells.

Lipardi C, Mora R, Colomer V, Paladino S, Nitsch L, Rodriguez-Boulan E, Zurzolo C - J. Cell Biol. (1998)

Bottom Line: Biol.However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts.Alternatively, cav1 and caveolae may not be directly involved in these processes.

View Article: PubMed Central - PubMed

Affiliation: Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy.

ABSTRACT
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI-anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI-anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42-53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI-anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.

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Purification of caveolin-enriched fractions on sucrose  density gradients in MDCK and cav1-transfected FRT cells (I).  cav1-FRT and MDCK cells were labeled with [35S]met-cys for 30  min and then chased for 3 h. Cells were lysed in TNE/TX-100  buffer and then run through a linear 5–40% sucrose gradient.  Fractions of 1 ml were collected from top to bottom after centrifugation to equilibrium, and then caveolin was immunoprecipitated from all fractions. After solubilization in Laemmli buffer  and boiling for 5 min, the samples were run on a 6–15% acrylamide gradient SDS gel. Caveolin is present in both the soluble  (9–12) and insoluble (5–7) fractions in both cav1-FRT and  MDCK cells.
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Figure 5: Purification of caveolin-enriched fractions on sucrose density gradients in MDCK and cav1-transfected FRT cells (I). cav1-FRT and MDCK cells were labeled with [35S]met-cys for 30 min and then chased for 3 h. Cells were lysed in TNE/TX-100 buffer and then run through a linear 5–40% sucrose gradient. Fractions of 1 ml were collected from top to bottom after centrifugation to equilibrium, and then caveolin was immunoprecipitated from all fractions. After solubilization in Laemmli buffer and boiling for 5 min, the samples were run on a 6–15% acrylamide gradient SDS gel. Caveolin is present in both the soluble (9–12) and insoluble (5–7) fractions in both cav1-FRT and MDCK cells.

Mentions: To rule out the possibility that the lack of effect of cav1 on the partitioning of gD1–DAF in TIFF was because of the inability of caveolin itself to localize in the insoluble fractions in the transfected FRT clones, we performed a sucrose density–gradient purification of TIFF followed by immunoprecipitation of the different fractions with an anticaveolin antibody. We found that a considerable amount of the protein (20–30% of the total) was found in the TIFF (fractions 5, 6, and 7 corresponding to 20–25% sucrose; Fig. 5, top). As a control we also followed the migration of caveolin to the TIFF in MDCK cells. In this cell line, a similar amount of the protein migrated to the lighter fractions 5, 6, and 7 of the gradient (Fig. 5, bottom). Therefore, the lack of effect of caveolin on GPI–proteins/GSLs cluster formation was not because of the inability of caveolin itself to localize in the TIFF.


Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells.

Lipardi C, Mora R, Colomer V, Paladino S, Nitsch L, Rodriguez-Boulan E, Zurzolo C - J. Cell Biol. (1998)

Purification of caveolin-enriched fractions on sucrose  density gradients in MDCK and cav1-transfected FRT cells (I).  cav1-FRT and MDCK cells were labeled with [35S]met-cys for 30  min and then chased for 3 h. Cells were lysed in TNE/TX-100  buffer and then run through a linear 5–40% sucrose gradient.  Fractions of 1 ml were collected from top to bottom after centrifugation to equilibrium, and then caveolin was immunoprecipitated from all fractions. After solubilization in Laemmli buffer  and boiling for 5 min, the samples were run on a 6–15% acrylamide gradient SDS gel. Caveolin is present in both the soluble  (9–12) and insoluble (5–7) fractions in both cav1-FRT and  MDCK cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140173&req=5

Figure 5: Purification of caveolin-enriched fractions on sucrose density gradients in MDCK and cav1-transfected FRT cells (I). cav1-FRT and MDCK cells were labeled with [35S]met-cys for 30 min and then chased for 3 h. Cells were lysed in TNE/TX-100 buffer and then run through a linear 5–40% sucrose gradient. Fractions of 1 ml were collected from top to bottom after centrifugation to equilibrium, and then caveolin was immunoprecipitated from all fractions. After solubilization in Laemmli buffer and boiling for 5 min, the samples were run on a 6–15% acrylamide gradient SDS gel. Caveolin is present in both the soluble (9–12) and insoluble (5–7) fractions in both cav1-FRT and MDCK cells.
Mentions: To rule out the possibility that the lack of effect of cav1 on the partitioning of gD1–DAF in TIFF was because of the inability of caveolin itself to localize in the insoluble fractions in the transfected FRT clones, we performed a sucrose density–gradient purification of TIFF followed by immunoprecipitation of the different fractions with an anticaveolin antibody. We found that a considerable amount of the protein (20–30% of the total) was found in the TIFF (fractions 5, 6, and 7 corresponding to 20–25% sucrose; Fig. 5, top). As a control we also followed the migration of caveolin to the TIFF in MDCK cells. In this cell line, a similar amount of the protein migrated to the lighter fractions 5, 6, and 7 of the gradient (Fig. 5, bottom). Therefore, the lack of effect of caveolin on GPI–proteins/GSLs cluster formation was not because of the inability of caveolin itself to localize in the TIFF.

Bottom Line: Biol.However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts.Alternatively, cav1 and caveolae may not be directly involved in these processes.

View Article: PubMed Central - PubMed

Affiliation: Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy.

ABSTRACT
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI-anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI-anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42-53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI-anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.

Show MeSH
Related in: MedlinePlus