Endocytosis of GPI-linked membrane folate receptor-α

Abstract

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 α in KB-cells. MFR-α was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-α was completely soluble in Triton X-100 at 0°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°C or in the presence of saponin at 0°C. Similar solubilization characteristics were found when MFR-α at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand, Triton X-100-insoluble membrane domains containing MFR-α could be separated from soluble MFR-α on sucrose flotation gradients. Only Triton X- 100 soluble MFR-α 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-α recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0°C. Using immunoelectron microscopy, we found MFR-α 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-α bypasses caveolae.