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.

Abstract

Chinese hamster ovary cells (CHO-Kl) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-a.-amino isobutyric acid (MeAIB, a nonmetabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid transinhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-Kl pro-cells by preventing proline transport, and alanine-resistant mutants (alar have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results.The A system of amino acid transport is Na+ dependent and transports mainly the small, polar, straight-chain amino acids such as alanine, the non-metabolizable analogs 2-aminoisobutyric acid and N-methyl-oa-amino isobutyric acid (MeAIB), and also proline. 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). In general, amino acids that are transported through the A system have been shown to be the most effective in the repression of this system (12). However, this specificity has been disputed (13). When derepressed cells are given an A system amino acid, a rapid decline in A system activity occurs (9-13). With some cell lines, a ...

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.

“…Effect of Derepression on A System Activity and the Na',K+-ATPase. We have previously shown that starving CHO-Ki cells for amino acids that are generally transported by the A system resulted in an increase in A system activity under conditions that required both mRNA and protein synthesis (7). Amino acids that were generally transported by this system, such as proline and MeAIB, acted as co-ri inhibiting the increase in A system activity and associated mRNA.…”

“…CHO-K1 (WT), alar4, and WT-alar4 hybrid H3, as well as the medium used in this study, MEMCHO-4, and the culture conditions used have been described (7). Hybrid H4 was isolated previously (7) but was not analyzed at that time.…”

“…The ri is believed to act by inhibiting transcription of the mRNA for the A system transporter and also, probably indirectly, inactivating the transporter itself (7). This model has been modified to incorporate our recent findings, suggesting that the A system and the Na+,K+-ATPase are coordinately regulated by gene R1 (Fig.…”

“…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).…”

Up‐regulation of system A activity in the regenerating rat liver

“…It is regulated in vivo and in vitro by both hormonal and nutritional factors [l-3]. System A expression is also regulated at the transcriptional level in cultured cells by amino acid availability, an effect which is called adaptive regulation and involves the de-repression of system A by a putative regulatory gene [4,5]. Also, anisotonic shock seems to selectively induce system A by protein s~thesis-dependent mechanisms which seem to occur through pathways other than the ones involved in adaptive regulation [6,7].…”