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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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Steady-state distribution of endogenous GPI-anchored proteins in cav1-transfected FRT cells. FRT  cells expressing cav1, grown  to confluence on polycarbonate filters for 5 or 6 d, were  subjected to domain-specific  biotinylation from the apical  (lanes A−, A+) or from the  basolateral (lanes B−, B+)  surface of the monolayer. After extraction with Triton  X-114 and phase separation,  detergent phases were incubated in the presence (lanes  A+, B+) or in the absence  (lanes A−, B−) of GPI-specific phospholipase C (6 U/ ml). After phase separation, biotinylated proteins in the aqueous  phase were TCA precipitated, subjected to SDS-PAGE, and then  visualized by Western blotting using 125I-streptavidin. Molecular  masses (top to bottom, are 116.5, 80, 49.5, 32.5, and 27.5 kD). In  cav1-FRT cells, one GPI-anchored protein is not polarized,  whereas the rest are found on the basolateral membrane, as was  previously shown in nontransfected FRT cells (Zurzolo et al., 1993).
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Figure 7: Steady-state distribution of endogenous GPI-anchored proteins in cav1-transfected FRT cells. FRT cells expressing cav1, grown to confluence on polycarbonate filters for 5 or 6 d, were subjected to domain-specific biotinylation from the apical (lanes A−, A+) or from the basolateral (lanes B−, B+) surface of the monolayer. After extraction with Triton X-114 and phase separation, detergent phases were incubated in the presence (lanes A+, B+) or in the absence (lanes A−, B−) of GPI-specific phospholipase C (6 U/ ml). After phase separation, biotinylated proteins in the aqueous phase were TCA precipitated, subjected to SDS-PAGE, and then visualized by Western blotting using 125I-streptavidin. Molecular masses (top to bottom, are 116.5, 80, 49.5, 32.5, and 27.5 kD). In cav1-FRT cells, one GPI-anchored protein is not polarized, whereas the rest are found on the basolateral membrane, as was previously shown in nontransfected FRT cells (Zurzolo et al., 1993).

Mentions: To determine whether caveolin transfection affected the polarity of endogenous GPI-anchored proteins in FRT cells, we studied their steady-state localization using domain-selective biotinylation in combination with partition into Triton X-114 and treatment with phospholipase C (Lisanti et al., 1988). As shown in Fig. 7, the majority of endogenous GPI-anchored proteins were basolaterally localized in the cav1-expressing cells. This result was identical to that obtained in nontransfected cells (Zurzolo et al., 1993). Therefore, caveolin expression appears to have no effect on GPI-anchored protein distribution, which remains predominantly basolateral, exactly as in FRT cells not expressing caveolin.


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)

Steady-state distribution of endogenous GPI-anchored proteins in cav1-transfected FRT cells. FRT  cells expressing cav1, grown  to confluence on polycarbonate filters for 5 or 6 d, were  subjected to domain-specific  biotinylation from the apical  (lanes A−, A+) or from the  basolateral (lanes B−, B+)  surface of the monolayer. After extraction with Triton  X-114 and phase separation,  detergent phases were incubated in the presence (lanes  A+, B+) or in the absence  (lanes A−, B−) of GPI-specific phospholipase C (6 U/ ml). After phase separation, biotinylated proteins in the aqueous  phase were TCA precipitated, subjected to SDS-PAGE, and then  visualized by Western blotting using 125I-streptavidin. Molecular  masses (top to bottom, are 116.5, 80, 49.5, 32.5, and 27.5 kD). In  cav1-FRT cells, one GPI-anchored protein is not polarized,  whereas the rest are found on the basolateral membrane, as was  previously shown in nontransfected FRT cells (Zurzolo et al., 1993).
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Related In: Results  -  Collection

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Figure 7: Steady-state distribution of endogenous GPI-anchored proteins in cav1-transfected FRT cells. FRT cells expressing cav1, grown to confluence on polycarbonate filters for 5 or 6 d, were subjected to domain-specific biotinylation from the apical (lanes A−, A+) or from the basolateral (lanes B−, B+) surface of the monolayer. After extraction with Triton X-114 and phase separation, detergent phases were incubated in the presence (lanes A+, B+) or in the absence (lanes A−, B−) of GPI-specific phospholipase C (6 U/ ml). After phase separation, biotinylated proteins in the aqueous phase were TCA precipitated, subjected to SDS-PAGE, and then visualized by Western blotting using 125I-streptavidin. Molecular masses (top to bottom, are 116.5, 80, 49.5, 32.5, and 27.5 kD). In cav1-FRT cells, one GPI-anchored protein is not polarized, whereas the rest are found on the basolateral membrane, as was previously shown in nontransfected FRT cells (Zurzolo et al., 1993).
Mentions: To determine whether caveolin transfection affected the polarity of endogenous GPI-anchored proteins in FRT cells, we studied their steady-state localization using domain-selective biotinylation in combination with partition into Triton X-114 and treatment with phospholipase C (Lisanti et al., 1988). As shown in Fig. 7, the majority of endogenous GPI-anchored proteins were basolaterally localized in the cav1-expressing cells. This result was identical to that obtained in nontransfected cells (Zurzolo et al., 1993). Therefore, caveolin expression appears to have no effect on GPI-anchored protein distribution, which remains predominantly basolateral, exactly as in FRT cells not expressing caveolin.

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