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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 gD1–DAF-enriched fractions on sucrose  density gradients. FRT cells expressing gD1–DAF and caveolin  (cl1 and cl2) 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 gD1–DAF was immunoprecipitated from all fractions. In both clones, mature and immature gD1–DAF forms are  almost exclusively restricted to the bottom fractions.
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Figure 4: Purification of gD1–DAF-enriched fractions on sucrose density gradients. FRT cells expressing gD1–DAF and caveolin (cl1 and cl2) 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 gD1–DAF was immunoprecipitated from all fractions. In both clones, mature and immature gD1–DAF forms are almost exclusively restricted to the bottom fractions.

Mentions: We then analyzed the TX-100–insoluble fractions by sucrose density–gradient centrifugation and immunoprecipitation of gD1–DAF from all fractions. In the caveolin- expressing FRT clones, neither the mature nor immature forms of gD1–DAF floated to the top of the gradient, as was shown in MDCK cells (Zurzolo et al., 1994); instead, both forms were enriched in fractions 8–12 (40% sucrose) at the bottom of the gradient, as expected for soluble proteins (Fig. 4). The small amount of gD1–DAF floating to TIFF in clone 2 was reproducible and corresponded to the small quantity of insoluble gD1–DAF found in this clone (Fig. 3). This result was not related to different levels of caveolin expressed by the two clones, but is simply a consequence of clonal variation among FRT cells. In fact, similar amounts of insoluble and floating gD1–DAF were found in the nontransfected FRT population lacking caveolin, as was previously reported (Zurzolo et al., 1994). These experiments indicated that transfection of cav1 was not able to reverse the inability of gD1–DAF to partition with TIFF in FRT cells.


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 gD1–DAF-enriched fractions on sucrose  density gradients. FRT cells expressing gD1–DAF and caveolin  (cl1 and cl2) 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 gD1–DAF was immunoprecipitated from all fractions. In both clones, mature and immature gD1–DAF forms are  almost exclusively restricted to the bottom fractions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Purification of gD1–DAF-enriched fractions on sucrose density gradients. FRT cells expressing gD1–DAF and caveolin (cl1 and cl2) 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 gD1–DAF was immunoprecipitated from all fractions. In both clones, mature and immature gD1–DAF forms are almost exclusively restricted to the bottom fractions.
Mentions: We then analyzed the TX-100–insoluble fractions by sucrose density–gradient centrifugation and immunoprecipitation of gD1–DAF from all fractions. In the caveolin- expressing FRT clones, neither the mature nor immature forms of gD1–DAF floated to the top of the gradient, as was shown in MDCK cells (Zurzolo et al., 1994); instead, both forms were enriched in fractions 8–12 (40% sucrose) at the bottom of the gradient, as expected for soluble proteins (Fig. 4). The small amount of gD1–DAF floating to TIFF in clone 2 was reproducible and corresponded to the small quantity of insoluble gD1–DAF found in this clone (Fig. 3). This result was not related to different levels of caveolin expressed by the two clones, but is simply a consequence of clonal variation among FRT cells. In fact, similar amounts of insoluble and floating gD1–DAF were found in the nontransfected FRT population lacking caveolin, as was previously reported (Zurzolo et al., 1994). These experiments indicated that transfection of cav1 was not able to reverse the inability of gD1–DAF to partition with TIFF in FRT cells.

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