Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L.
The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.
Collarini
E J
EJ
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Campbell
G S
GS
Oxender
D L
DL
D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000235:genetics; D009154:Mutation; D010053:Ovary
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D002352:Carrier Proteins; D007935:Leucine-tRNA Ligase