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

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.

Figure 1

Localization by double immunofluorescence of cav1 and gD1–DAF in transfected FRT cells. A stable FRT clonal line expressing cav1 and gD1–DAF was grown to confluence on glass coverslips. Cells were fixed with paraformaldehyde and permeabilized with PBS CM containing 0.2% gelatin and 0.075% saponin, and then stained using a polyclonal antibody against caveolin (A) and a monoclonal antibody against gD1–DAF (B). Primary antibodies were visualized using anti–mouse TRITC-conjugated and anti-rabbit FITC-conjugated antibodies. Cav1 gave a punctate staining localized both at the plasma membrane and, less intensely, at the Golgi apparatus, whereas gD1–DAF was enriched mainly at the basolateral surface. Bar, 50 mm.

Figure 2

Surface distribution of gD1–DAF in cav1-transfected and wild-type FRT cells. FRT cells expressing gD1–DAF or both gD1– DAF and caveolin (cl1 and cl2) were grown on filters for 5 d and then labeled with [³⁵S]met-cys overnight. (A) Surface-expressed gD1–DAF was selectively biotinylated from the apical (A) or basolateral (B) side, extracted, and immunoprecipitated with a specific monoclonal antibody, and then the biotinylated fraction was subsequently precipitated with streptavidin beads. Immunoprecipitated proteins were visualized by 10% SDS-PAGE and autoradiography. Note that gD1–DAF was enriched on the basolateral surface (∼95% of total membrane proteins) both in transfected and nontransfected cells. (B) The quantity of caveolin synthesized by the two FRT clones was compared to the amounts present in MDCK cells by immunoprecipitation of [³⁵S]met-cys cells with a polyclonal antibody against caveolin.

Figure 3

Pulse-chase analysis of gD1–DAF solubility in TX-100 in FRT cells expressing cav1. FRT cells stably expressing gD1– DAF and caveolin (cl1 and cl2) were grown to confluence and then pulsed for 5 min with [³⁵S] met-cys, followed by incubation in chase medium for the indicated times. After extraction in TNE-1% TX-100 buffer at 4°C, both the soluble (S, supernatant) and insoluble (I, pellet) fractions were collected after centrifugation, and then gD1–DAF was subsequently immunoprecipitated and analyzed by SDS-PAGE and fluorography. In both clones, gD1–DAF is mostly soluble at all chase times during its transport to the cell surface. The small amount of insoluble gD1–DAF found in clone 2 is likely to be a result of clonal variation. In fact, a similar amount of insoluble gD1–DAF was also found in nontransfected cells (Zurzolo et al., 1994).

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.

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.

Figure 6

Purification of caveolin-enriched fractions on sucrose density gradients in MDCK and cav1-transfected FRT cells (II). Cav1-FRT and MDCK cells were treated as described in Fig. 5. 1-ml fractions from a 5–40% linear sucrose density gradient were immunoprecipitated using a polyclonal antibody against caveolin. Samples were lysed in Laemmli buffer and then left 30 min at 25°C before loading them on a 6–15% acrylamide gradient SDS gel. In both cav1-FRT and MDCK cells, caveolin forms oligomers of high molecular weight (∼350, 300, and 200 kD), which are localized in the bottom fractions as well as within the lighter fractions of the gradients.

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).

Figure 8

Electron micrographs of wild-type and caveolin-expressing FRT cells. Cav1-transfected and nontransfected FRT cells were grown on filters for 5 d, and then fixed and treated for EM as described in Materials and Methods. Apical side (A) and basal side (B) of nontransfected FRT cells. Note the absence of plasmalemmal caveolae on both surfaces. FRT cells transfected with cav1 contain caveolae both on the apical (C) and basolateral membranes (D and E) characterized as coat-free flask-shaped plasmalemmal invaginations with the diaphragm at the neck (small arrows). Note on the basal side (E) the characteristic cluster of caveolae into racemose structures (small arrow). cp indicates coated pit; n indicates nucleus; arrowheads show the edge of the filter.

Figure 9

Immunogold localization of gD1–DAF on the surface of caveolin-expressing FRT cells. Cav1-FRT cells were grown to subconfluence on coverslips, incubated with an anti-gD1–DAF monoclonal antibody on ice for 1 h and, after washing in PBS, with a secondary antibody conjugated with 10-nm colloidal gold on ice for 1 h. After fixation, cells were dehydrated through graded ethanol and were scraped from the coverslips in 70% ethanol. The pellets were then processed as described in Materials and Methods. A–D show different sections of apical and basolateral plasma membranes of cav1-FRT cells that contain gD1–DAF clustered in newly formed (A and B) or classical flask-shaped caveolae (C and D; small arrows). A racemose cluster of caveolae containing gD1–DAF is shown on the apical surface (D; large arrow). Arrowheads indicate the gold particles bound to the anti-gD1–DAF antibody. Bar, 100 nm.