SORTING AND INTRACELLULAR TRAFFICKING OF A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN AND 2 HYBRID TRANSMEMBRANE PROTEINS WITH THE SAME ECTODOMAIN IN MADIN-DARBY CANINE KIDNEY EPITHELIAL-CELLS

We compared the trafficking of the glycosylphosphatidylinositol (GPI)-anchored placental alkaline phosphatase (FLAP) and two chimeric transmembrane proteins containing the FLAP ectodomain in stably transfected Madin-Darby canine kidney epithelial cells to determine whether different mechanisms might be used in apical sorting of GPI-anchored and transmembrane proteins. PLAP-G, which contained the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein, was delivered directly to the basolateral surface. FLAP-HA contained the transmembrane and cytoplasmic domains of influenza hemagglutinin. Both FLAP and FLAP-HA were delivered directly to the apical membrane. FLAP becomes insoluble in Triton X-100 during biosynthetic transport, as it associates with detergent-resistant membranes. Neither hybrid protein was detergent insoluble, though the small amount of FLAP that was missorted to the basolateral surface was insoluble. We examined the effects of three drugs known to interfere with membrane trafficking on sorting and delivery of FLAP and the hybrid proteins. Monensin had no effect on sorting or surface expression of any of the proteins. Nocodazole affected the sorting of both FLAP and FLAP-HA but not of PLAP-G. Brefeldin A appeared to disrupt the sorting of FLAP and FLAP-HA but not of PLAP-G. This conclusion was tempered by the observation that this drug affected the distribution of proteins at the cell surface. Thus, sorting and transport of GPI-anchored and apical transmembrane proteins are similar in a number of respects.