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Endocytosis of GPI-linked membrane folate receptor-alpha
S Rijnboutt1, G Jansen, G Posthuma
1Department of Cell Biology, Faculty of Medicine, Universiteit Utrecht, The Netherlands.
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 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.
Insights
Membrane folate receptors (MFRs) are internalized via Triton X-100 soluble pathways, bypassing caveolae. Internalized MFR-alpha recycles slowly to the cell surface, remaining soluble.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Glycosylphosphatidylinositol (GPI)-linked membrane folate receptors (MFRs) mediate cellular uptake of folates and related drugs.
- Understanding the transport and internalization mechanisms of MFRs is crucial for drug delivery and folate metabolism research.
Purpose of the Study:
- To investigate the biosynthetic transport and internalization pathways of MFR isoform alpha in KB cells.
- To determine the role of membrane microdomains in MFR-alpha internalization and trafficking.
Main Methods:
- Metabolic labeling and pulse-chase experiments to track MFR-alpha synthesis and maturation.
- Differential solubilization with Triton X-100 and saponin to analyze membrane association.
- Sucrose flotation gradients to separate membrane domains.
- Ligand binding studies using a photoaffinity analog of folic acid.
- Immunoelectron microscopy to visualize MFR-alpha localization.
Main Results:
- MFR-alpha matures from a 32-kD soluble form to a 36-38 kD glycosylated species.
- Mature MFR-alpha exhibits differential solubility in Triton X-100, indicating association with detergent-resistant membrane domains.
- Only Triton X-100 soluble MFR-alpha is internalized from the plasma membrane.
- Internalized MFR-alpha recycles slowly to the cell surface in a soluble state.
- MFR-alpha is found throughout the endocytic pathway, primarily in clathrin-coated vesicles and endosomes, suggesting caveolae-independent internalization.
Conclusions:
- Internalization of MFR-alpha is dependent on its solubility in Triton X-100, suggesting a pathway distinct from detergent-resistant membrane domains.
- The reduced-folate-carrier is excluded from detergent-resistant domains, indicating distinct membrane localization.
- Internalizing MFR-alpha largely bypasses caveolae, utilizing clathrin-mediated endocytosis.
- Recycled MFR-alpha remains in a soluble state, facilitating further rounds of folate uptake.