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

A mutated yeast cell 22574d lacking all three proline transporters, PUT4, U GA4, and GAP1, and incapable of growth on proline recovers its lost ability to grow on proline as sole nitrogen source when transformed with a mutagen ized mouse gamma -actin cDNA (M-gamma -A). Native mouse gamma -actin cDNA i s ineffective. The 3'-region of gamma -actin cDNA was mutagenized to resemb le 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. Af ter 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. White the transformation with M-gamma -A increased th e uptake of both proline and gamma -amino butyric acid, the transport of fi ve 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 o f an mRNA for UGA4 in both transformed and nontransformed cells but no evid ence for the expression of CAP1 or PUT4. The mRNA for UCA4 was expressed at a lower level in strain 22574d than in the parent yeast Sigma 1278b. Furth ermore, the message in the mutated cells is smaller in size by about 15%. T hese results are consistent with the synthesis of a mutated transporter whi ch requires the coexpression of M-gamma -A, but not native gamma -actin, to restore physiological function, i.e., proline or gamma -amino acid transpo rt. J. Cell. Physiol. 186:124-135, 2001. (C) 2001 Wiley-Liss, Inc.