Detergent-insoluble GPI-anchored proteins are apically sorted in Fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting

In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells d eliver the majority of endogenous,glycosylphosphatidyl inositol (GPI)-ancho red proteins to the basolateral surface. However, we report here that the G PI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor -Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transf ected Fischer rat thyroid cells. In agreement with the "raft hypothesis," w hich postulates the incorporation of GPI proteins into,glycosphingolipids a nd cholesterol-enriched rafts, we found that both of these proteins were in soluble in Triton X-100 and floated into the lighter fractions of sucrose d ensity gradients. However, disruption of lipid rafts by removal of choleste rol did not cause surface missorting of FLAP and NTR-LAP, and the altered. surface sorting of these proteins after Fumonisin B1 treatment did not corr elate with reduced levels in Triton X-100 -insoluble fractions. Furthermore , in contrast to the GPI-anchored forms of both of these proteins, the secr etory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid mi crodomains. Together, these data demonstrate that the GPI anchor is require d to mediate raft association but is not sufficient to determine apical sor ting. They also suggest that signals present in the ectodomain of the prote ins play a major role and that lipid rafts may facilitate the recognition o f these signals in the trans-Golgi network, even though they are not requir ed for epical sorting.