Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems

Abstract

We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CHO-K1 pro- cells. Mutants alar2 and alar3, isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro-, stable, cross resistant to alpha-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake. Ethyl methane sulfonate (EMS) increases the frequency of pro- alar clones in the population by at least 50 times the spontaneous frequency. The increased velocity of proline transport by alar2 and alar3 can be attributable to the 1.5 to 3 times increase in velocity of transport of proline through systems A, ASC, and P. The Vmax for proline transport through the A system has increased two times for alar2 while the Km and Vmax for alar3 has increased by 1.4 and 2.3 times that of CHO-K1. There is a corresponding increase in Vmax of proline transport by alar2 through the P system. The P system is defined operationally as that portion of the Na+-dependent velocity that remains when the A, ASC, and glutamine-inhibitable fraction are eliminated. The system is concentrative. Proline appears to be the preferred substrate. Li+ cannot be substituted for Na+. The system is moderately dependent upon pH. It obeys Michaelis-Menten kinetics and is not derepressible by starvation. There is no evidence for an N system in CHO-K1.

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

“…It is Na' dependent and transports one Na' into the cells with each amino acid (3). This system is inducible by hormones (4)(5)(6), repressible by amino acids, which it generally transports (7)(8)(9)(10)(11), and present with increased activity in many transformed and malignant cells (6).…”

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

“…This system is ubiquitous among the vertebrates (6,14,22,26,28; see references in 2, 3, and 12). Of the three Na+-dependent transport systems (A, ASC, and P) present in Chinese hamster ovary cells, CHO-Kl, the cell line with which we will be concerned, the A system, appears to be the only one whose activity is repressible (20).When cells are placed under conditions in which they are starved for amino acids, a severalfold increase in velocity (Vmax) of transport of amino acids through the A system occurs. This increase has been shown to be inhibited by cycloheximide and actinomycin D (9-13, 15).…”

“…This system is ubiquitous among the vertebrates (6,14,22,26,28; see references in 2, 3, and 12). Of the three Na+-dependent transport systems (A, ASC, and P) present in Chinese hamster ovary cells, CHO-Kl, the cell line with which we will be concerned, the A system, appears to be the only one whose activity is repressible (20).…”

“…It has been shown previously that incubation of CHO-Ki pro-cells under starvation conditions results in a severalfold increase in the velocity of proline transport through the A system (20). Utilizing the antagonism between proline and other A system amino acids on the growth of CHO-Kl pro-cells (4), a selection system has been developed for the isolation of mutants that have elevated levels of amino acid transport (20).…”

“…All cell lines were routinely grown as monolayers in MEMCHO-4 as previously described (20). MEMCHO-4 hypoxanthine-aminopterin-thymidine medium containing 1 mM ouabain was employed in hybrid selection.…”