FIGURE 7.

Expression of recombinant rat MTHFD2L in yeast. A, S. cerevisiae strain MWY4.4 (ser1 ura3 trp1 leu2 his4 ade3-65 Δmtd1) harboring YEp-rMTHFD2L and untransformed control cells were grown in selective medium, harvested, and lysed as described under “Experimental Procedures.” Lysates were assayed for NAD⁺- and NADP⁺-dependent CH₂-THF dehydrogenase activity before (whole lysate) and after a 30,000 × g centrifugation (cleared lysate). Enzyme activity is expressed as nmol product per gm of wet weight (nmol/gm wet wt) of cells. Each column represents the average ± S.D. of duplicate determinations. B, strain MWY4.5 (ser1 ura3 trp1 leu2 his4 ade3-30/65 Δmtd1) was transformed to uracil prototrophy with either YEp-rMTHFD2L or empty plasmid (Yep24ES). Ura⁺ transformants were streaked onto yeast minimal plates containing serine (left) or serine + adenine (Ser + Ade, right) and incubated at 30 °C for 4 days. Both plates also contained leucine, tryptophan, and histidine to support the other auxotrophic requirements of MWY4.5.