Two membrane-bound proteins associated with alanine resistance and increased A-system amino acid transport in mutants of CHO-K1.
Growth of CHO-K1, a proline auxotroph, is inhibited by amino acids that prevent proline transport. From a hydroxyurea-treated, alanine-resistant, constitutive mutant, alar4, we isolated, in a stepwise fashion, mutants, resistant to higher concentrations of alanine, that have increased velocity of amino acid transport through the A system. Two such mutants, alar4-H2.1 and alar4-H3.9, isolated as resistant to 50 mM and 125 mM alanine, respectively, showed increases in Vmax of proline transport through the A system that are directly proportional to their resistance to alanine. Alar4-H3.9, as compared to alar4 and CHO-K1, has six and 29 times the Vmax of proline transport through the A system and two and five times the velocity of transport through the combined ASC and P systems, respectively, and no change in system L. No double-minute or homologous staining regions were detectable in alar4-H3.9. A-system activity of alar4-H2.1 and alar4-H3.9, when grown under nonselective conditions, was stable for 20 generations and then declined. The phenotype of alar4-H3.9 is codominant with that of alar4 and partially recessive to that of CHO-K1. Membrane vesicles prepared from alar4-H3.9 show increases mainly in A-system transport. In sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of A-system active membrane vesicles and endoplasmic reticulum, two bands of molecular weight of approximately 62-66 kd and 29 kd are present in higher concentrations in alar4-H3.9 than in CHO-K1. These results are compatible with the hypothesis that the phenotype of alar4-H3.9 is the result of gene amplification of an A-system transporter structural gene and that the two bands may represent this transporter.
Department of Biological Sciences, University of California-Santa Barbara 93106.
J
J
Moffett
E
E
Mendiaz
M
M
Jones
E
E
Englesberg
D000409:Alanine / Q000235:genetics*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D006918:Hydroxyurea / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation
D000596:Amino Acids; D008565:Membrane Proteins; D000409:Alanine; D006918:Hydroxyurea