Endocytosis of GPI-linked membrane folate receptor-alpha

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs, We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells, MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degrees C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degrees C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable I-125-labeled photoaffinity-analog of folic acid as a ligand, Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane, The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains, Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degrees C. Using immunoelectron microscopy, we found MFR-alpha: along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles, In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.