Mutated-?-actin restores growth to a yeast amino acid transport defective mutant

Abstract

A mutated yeast cell 22574d lacking all three proline transporters, PUT4, UGA4, and GAP1, and incapable of growth on proline recovers its lost ability to grow on proline as sole nitrogen source when transformed with a mutagenized mouse gamma-actin cDNA (M-gamma-A). Native mouse gamma-actin cDNA is ineffective. The 3'-region of gamma-actin cDNA was mutagenized to resemble E51 cDNA previously isolated from Ehrlich tumor cells. The E51 cDNA has an extended reading frame in the 3'-region compared to that in native gamma-actin. The extension of the open reading frame in E51 cDNA, was found to be due to an additional pair of bases (TG) at position 1104 of E51 cDNA. After site-directed mutagenesis of the 3'-region of native gamma-actin cDNA to resemble that of E51 cDNA, the construct, M-gamma-A cDNA, was expressed in the 22574d yeast. While the transformation with M-gamma-A increased the uptake of both proline and gamma-amino butyric acid, the transport of five other solutes was not changed by this transformation. Northern blotting of the nontransformed and the M-gamma-A-transformed 22574d cells with gene-specific probes for the three proline transporters showed the expression of an mRNA for UGA4 in both transformed and nontransformed cells but no evidence for the expression of GAP1 or PUT4. The mRNA for UGA4 was expressed at a lower level in strain 22574d than in the parent yeast sigma1278b. Furthermore, the message in the mutated cells is smaller in size by about 15%. These results are consistent with the synthesis of a mutated transporter which requires the coexpression of M-gamma-A, but not native gamma-actin, to restore physiological function, i.e., proline or gamma-amino acid transport.