Regulation and genetics of amino acid transport.

Abstract

The major transport systems for the uptake of neutral amino acids in mammalian cells have been designated A, ASC, and L.’,’ The transport systems and their regulation have been characterized in Chinese hamster ovary (CHO) System A is sodium-dependent, subject to trans-inhibition, and serves for the uptake of amino acids with short, polar, or linear side chains. System ASC is also sodium dependent and has a strong preference for alanine, serine, and cysteine. In the C H O cell, the ASC system shows a somewhat broader specificity than that found in the Ehrlich cell.* Unlike System A, System ASC does not tolerate N-methylated substrates such as 2-methylaminoisobutyric acid (MeAIB). System L is sodium-independent and serves for the uptake of branched-chain and aromatic amino acids. We operationally define the systems as follows: System A can be represented by the sodium-dependent uptake of 0.2 m M 2-arninoisobutyric acid (AIB) that is inhibited by 25 m M MeAIB; System ASC, the sodium-dependent uptake of 0.2 m M L-alanine that is not inhibited by 25 m M MeAIB; and System L, the sodium-independent uptake of 0.2 m M L-leucine that is inhibited by 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH).3 Although the systems have a preferred set of substrates, they do have overlapping specificities. FIGURE 1 shows the contributions of these systems to the uptake of individual amino acids in CHO-K1 cells. This overlap makes the study of transport systems in isolation difficult. The availability of mutations in one or more of the transport systems would greatly facilitate the study of the function and regulation of the transport systems. We are currently combining genetic approaches with kinetic studies using C H O cells because of the relative ease with which mutants can be obtained from these cells. Furthermore, C H O cells can be used to form interspecies hybrids with human cells.’ The hamster-human hybrid cells preferentially segregate the human chromosomes, permitting the assignment of a phenotype to a particular chromosome. In the present study, we have isolated and characterized C H O mutants defective in the regulation of System L6 and mutants with reduced System L transport activity. We have also used hamster-human hybrids to map System L transport activity to human chromosome 20.’