Membrane folate-binding proteins are responsible for folate-protein conjugate endocytosis into cultured cells.
Folate-protein conjugates have been shown to bind to and enter HeLa and KB cells by receptor-mediated endocytosis [Leamon and Low (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5572-5576]. Although these cells contain a membrane folate-binding protein (FBP) involved in the uptake of free folate, no studies have been conducted to evaluate whether the folate-protein conjugates enter cells via the same protein. To address this issue, HeLa cell monolayers were treated with folate-labelled 125I-RNAase under various conditions characteristic of FBP-mediated folate uptake. Folate-labelled 125I-RNAase was found to bind to cells with high affinity (Kd = 24 nM), and like the free vitamin, its binding could be competitively blocked by excess free folate. Furthermore, binding could be reversed by either washing the cells with acid/saline, pH 3.0, or by treating the cells with phosphatidyl-inositol-specific phospholipase C, an enzyme known to release FBP from cell surfaces. Because cells pretreated with anti-FBP serum were unable to bind folate conjugates, and since the same antiserum identified a single 65 kDa band reminiscent of FBPs found in many other tissues, we conclude that a classical FBP is responsible for the uptake of folate-protein conjugates by receptor-bearing cells.
Department of Chemistry, Purdue University, W. Lafayette, IN 47907-1393.
C P
CP
Leamon
P S
PS
Low
D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007457:Iodine Radioisotopes; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011956:Receptors, Cell Surface; D012260:Ribonucleases / Q000378:metabolism
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D007106:Immune Sera; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D005492:Folic Acid; D012260:Ribonucleases; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase