Intracellular trafficking/membrane targeting of human reduced folate carrier expressed in Xenopus oocytes

The major cellular pathway for uptake of the vitamin folic acid, including its absorption in the intestine, is via a plasma membrane carrier system, t he reduced folate carrier (RFC). Very little is known about the mechanisms that control intracellular trafficking and plasma membrane targeting of RFC . To begin addressing these issues, we used Xenopus oocyte as a model syste m and examined whether the signal that targets the protein to the plasma me mbrane is located in the COOH-terminal cytoplasmic tail or in the backbone of the polypeptide. We also examined the role of microtubules and microfila ments in intracellular trafficking of the protein. Confocal imaging of huma n RFC (hRFC) fused to the enhanced green fluorescent protein (hRFC-EGFP) sh owed that the protein was expressed at the plasma membrane, with expression confined almost entirely to the animal pole of the oocyte. Localization of hRFC at the plasma membrane was not affected by partial or total truncatio n of the COOH-terminal tail of the polypeptide, whereas a construct of the cytoplasmic tail fused to EGFP was not found at the plasma membrane. Disrup tion of microtubules, but not microfilaments, prevented hRFC expression at the plasma membrane. These results demonstrate that the molecular determina nt(s) that directs plasma membrane targeting of hRFC is located within the backbone of the polypeptide and that intact microtubules, but not microfila ments, are essential for intracellular trafficking of the protein.