Tritium suicide selection of mammalian cell mutants defective in the transport of neutral amino acids

Abstract

Mouse lymphocytic cells of the established line GF-14 were allowed to accumulate intracellular 3H-labeled aminoisobutyric acid (AIB), frozen, and stored over liquid N2. After internal radiation had reduced survival to 1 in 10(4), survivors were plated and tested for their ability to transport AIB. Out of 200 clones tested, two (designated GF-17 and GF-18) were found to have reductions to 13-35% of the parent in the rate of transport of AIB, L-alanine, L-proline, and L-serine; GF-18 also showed significant reductions in the rate of transport of L-glutamate and DL-cysteine. Little or no change was observed for 10 other amino acids or for thymidine. Kinetic analyses revealed that the mutants were not altered in Km for AIB uptake, but had Vmax values approximately 20% the value of the parent strain, GF-14, suggesting that either the number of AIB transport sites or the turnover rate of the sites has been reduced in the two mutants.