Structural and functional properties of the folate transport protein from a methotrexate-resistant subline of Lactobacillus casei.
A methotrexate-resistant subline of Lactobacillus casei has been isolated which transports folate at a reduced rate and contains a binding protein whose affinity for folate (Kd = 280 nM) is considerably lower than that of the corresponding protein of wild-type cells (Kd = 0.6 nM). After the addition of mercaptoethanol, however, this same protein exhibits a high affinity for folate (Kd = 1.2 nM) and transports the substrate at a normal rate. Subsequent removal of mercaptoethanol causes a rapid reversal of the activation process. Binding protein labeled covalently with carbodiimide-activated [3H]folate, solubilized with Triton X-100, and subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate had an apparent molecular weight which was approximately twofold higher than that of the corresponding protein of wild-type cells, but it could be reduced to the parental size (Mr = 20,000) by prior treatment with mercaptoethanol. Purified binding protein also exhibited a similarly elevated molecular weight, and its amino acid composition was indistinguishable from that of the wild-type counterpart, except for the presence of a single cysteine residue. These findings indicate that the mutant binding protein exists in a low-affinity form due to disulfide bridge formation between two homologous protein subunits and that cleavage of this bond by mercaptoethanol generates the high-affinity state. The rapid and specific interconversion of these binding forms suggests further that the high-affinity form of the binding protein also resides in the membrane as a dimer, held together by noncovalent interactions.
D000596:Amino Acids / Q000032:analysis; D001692:Biological Transport; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008623:Mercaptoethanol / Q000494:pharmacology; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D011956:Receptors, Cell Surface; D013831:Thiamine / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D008623:Mercaptoethanol; D005492:Folic Acid; D005022:Ethyldimethylaminopropyl Carbodiimide; D013831:Thiamine; D008727:Methotrexate