EPITHELIAL SORTING OF A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED BACTERIAL PROTEIN EXPRESSED IN POLARIZED RENAL MDCK AND INTESTINAL CACO-2 CELLS

To evaluate whether a glycossylphosphatidylinositol (GPI) anchor can f unction as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-1 14 phase-partitioning and susceptibility to phosphoinositol-specific p hospholipase C. In polarized MDCK cells, EGE' was localized almost exc lusively to the apical felt surface, while in polarized intestinal Cac o-2 cells, although 80% of the extracellular form of the enzyme was ro uted through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotin ylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral, EGE' delivered to the basolateral cell surface w as transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal e pithelial cells. However, the mis-,sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-a nchored proteins.