FIGURE 7.

AdoHcy accumulates in the presence of homocysteine. A, growth of wild type YNM1(met25), and YNM5 (met25 met6 str4), and YNM6 (met6 str4) mutant strains on inositol- and choline-free plates in the presence and absence of 1 mm homocysteine. 5-μl dilutions of A₆₀₀ = 1, 0.1, 0.01, and 0.001 (black triangles) were spotted onto the plates. Images were taken after 2 days of growth. n.s., no supplementation. B, accumulation of d₄-AdoHcy by YNM5 (met25 met6 str4) mutant cells grown in the presence of d₄-Hcy. YNM5 (met25 met6 str4) mutant cells were pregrown in YPD medium and inoculated at A₆₀₀ = 0.025 in fresh inositol- and choline-free medium containing 1 mmd₄-Hcy, and after 15 h of growth (early log phase), AdoHcy and AdoMet were extracted and analyzed by HPLC-ESI-MS/MS (C-18 column, water-acetonitrile gradient, TSQ 7000 Triple-Quad MS (42)) as described under “Experimental Procedures.” AdoHcy, d₄-AdoHcy, AdoMet, and d₃-AdoMet were traced in the chromatograms by virtue of their characteristic m/z values, as indicated in the figure. C, relative AdoHcy content in the YNM5 (met25 met6 str4) mutant strain in the presence and absence of homocysteine. YNM5 mutant cells were pregrown in YPD medium and inoculated at A₆₀₀ = 0.025 in fresh inositol- and choline-free medium with or without 1 mm homocysteine supplementation. At the indicated time points AdoHcy was extracted and analyzed by HPLC-ESI-MS/MS as described under “Experimental Procedures.” Data from early log phase (15 h) are shown.