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 degree 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 degree C. Similar solubilization characteristics were found when MFR-alpha 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-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 degree 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.
Rijnboutt
S
S
Department of Cell Biology, Faculty of Medicine, Universiteit Utrecht, The Netherlands.
Jansen
G
G
Posthuma
G
G
Hynes
J B
JB
Schornagel
J H
JH
Strous
G J
GJ
D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004705:Endocytosis / Q000502:physiology*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives; D005493:Folic Acid Antagonists / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008024:Ligands; D016253:Microscopy, Immunoelectron; D017830:Octoxynol / Q000494:pharmacology; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D017261:Glycosylphosphatidylinositols; D008024:Ligands; D011956:Receptors, Cell Surface; D017830:Octoxynol; D005492:Folic Acid