alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

Abstract

A constitutive mutant, alar4, for the A system of amino acid transport, has increased activity and amount of the A system. This is accompanied by increased sensitivity to ouabain, as measured by efficiency of plating, and increased activity and abundance of the Na+,K+-ATPase that is present in the parental cell line, CHO-K1 (wild type). The latter was shown by increases in (i) ouabain-inhibitable "Rb uptake in intact cells, (ii) ouabain-inhibitable ATPase activity in mixed membrane vesicles, and (iii) number of ouabain-binding sites and by similar Kd values for ouabain binding and Ki12 for ouabain inhibition of Na+,K+-ATPase as compared to the wild type. The increase in abundance of the Na' pump is associated with a 4-fold increase in abundance of the mRNA for the al subunit of the Na+,K+-ATPase. We could not detect mRNA for a2 or a3 or for the .8 subunits. The increase in abundance of the A system and Na+,K+-ATPase is associated with a negligible increase in intracellular Na+ concentration. We propose that the increase in the abundance of the A system and the Na',K+-ATPase is the result of a mutation in regulatory gene RI that controls the A system and the Na',K+-ATPase and is not due to a primary effect of a possible initial increase in Na+ concentration.A study of a constitutive mutant of CHO-K1 cells, alar4, which has increased abundance of the membrane-bound transporter for the A system of amino acid transport (1-3), has revealed an interesting relationship between this system

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.

“…Since we could not detect any message for the subunit (see below) in our experiments, the possible effect of gene R1 on the expression of the gene for the f8 subunit remains to be determined. Although, as mentioned above, previous work had implicated an increase in intracellular Na' as being the causal factor triggering the increase in abundance of the Na',K+-ATPase, the fact that the intracellular Na' concentration in mutant alar4 is similar to that of the WT placed doubt on this proposed relationship (1).…”

“…1 starvation or in the absence of A system amino acids). This increase in A system activity is associated, in alar4, with an increase in abundance of the Na' ,K+-ATPase and mRNA for the a, subunit of this enzyme (1). The A system transporter in alar4 is not inactivated by A system amino acids as it is in the WT and this mutant is not further derepressible when incubated in amino acid-free medium.…”

“…Evidence has been presented suggesting that the activity of the A system of amino acid transport and the Na+,K+-ATPase is coordinately regulated at the transcriptional level by means of repression and negative control by the A system regulatory gene R1 (1).…”

Evidence for a regulatory protein involved in the increased activity of system A for neutral amino acid transport in osmotically stressed mammalian cells.

“…CHO-K1 mutants that exhibit increases in system A activity and enhanced expression of Na+,K+-ATPase (26,35) overexpress the protein P1 (hsp60) (36). Genetic analysis suggests that both transporters, system A and the sodium pump, are under coordinate control by regulatory gene RI (26,35).…”

“…Genetic analysis suggests that both transporters, system A and the sodium pump, are under coordinate control by regulatory gene RI (26,35). Overexpression of the three proteins may be the result of a mutation in a common regulatory element, the R1 product.…”