Isolation And Characterization Of Chinese Hamster Ovary Cell Mutants Defective In The Activity And The Regulation Of Amino Acid Transport System L.

Abstract

Our laboratory has been using genetic approaches to study the neutral amino acid transport System L in Chinese hamster ovary (CHO) cells. The regulation of System L has been characterized using the temperature-sensitive leucyl-tRNA synthetase mutant cell line, CHO-tsH1. When incubated at 39(DEGREES)C, these cells are starved for leucine because of their inability to charge leucyl-tRNA. Under these conditions, the activity of System L, which serves for the uptake of leucine, increases. We have isolated mutants defective in the regulation of System L transport activity by selecting temperature-resistant cells from mutagenized CHO-tsH1 cells. These mutants have constitutively elevated transport activity and increased intracellular steady-state levels of System L amino acids, which serve to complement the synthetase defect. Hybridization of one of the regulatory mutants with cells that show normal regulation of System L activity resulted in hybrid cells that are able to regulate leucine transport activity. These results suggest that a transacting element may be involved in the regulation of System L, and that this factor is defective or absent in the regulatory mutant. The increased transport activity of the regulatory mutants makes them more sensitive to toxic substrates. We subjected one of the regulatory mutant cell lines to a tritium-suicide selection, in which tritiated leucine served as a toxic substrate, and isolated mutants that have a specific reduction in System L transport activity. The reduction in transport activity leads to reduced intracellular steady-state levels of System L amino acids. In contrast to the parental cells, these cells are temperature sensitive because their reduced transport activity does not complement the defective synthetase. Since the reduction of System L transport activity limits the growth of these cells at 39(DEGREES)C, they can be used to clone the genes for System L transport by selecting for complementation of the transport defect.