Figure 5.
The AltPW functionally complements the yeast sah1 mutants. A, expression of N-terminally tagged GST-Pfs and GST-LuxS fusion proteins in yeast. Shown are heterozygous diploid sah1/SAH1 met25/MET25 transformants carrying the empty pYEX4T-1 or pYEX-4T-1 [Pfs/LuxS] plasmid containing E. coli pfs and luxS genes under the regulation of the CUP1p promoter. B, the AltPW suppresses the growth defect of the sah1 mutant. Wildtype yeast and the sah1 mutant carrying pYEX4T-1 or pYEX-4T-1 [Pfs/LuxS] plasmid were analyzed for growth on SD media with or without 0.05 mm CuSO4. Images were taken after 2 days of growth at 30 °C. C, the AltPW suppresses elevated AdoHcy levels in the sah1 mutant and in Hcy-supplemented wildtype yeast. Logarithmic-phase wildtype and the sah1 mutant yeast cells carrying pYEX4T-1 or pYEX-4T-1 [Pfs/LuxS] plasmid were cultivated in SD−Ura with or without 0.05 mm CuSO4 and 1 mm Hcy for 6 h before AdoHcy extraction. AdoHcy levels only for strains grown in the absence of additional CuSO4 supplementation are shown. AdoHcy was normalized to ISTD. Data are mean ± S.D. (error bars) from three independent experiments (***, p < 0.001).