Figure 5.

Human TPI_Ile170Val and TPI_Ile170Thr complement for yeast TPI and are catalytically active. (a) TPI, TPI_Ile170Val and TPI_Ile170Thr, but not TPI_Lys13Arg, complement for yeast TPI1. In a plasmid shuffle experiment, Δtpi1 cells carrying a counterselectable TPI-encoding plasmid were transformed with a centromeric plasmid (minichromosome) encoding the indicated TPI mutants. Transformed cells were then transferred to 5'FOA to induce loss of the counterselectable plasmid. Only cells containing a functional TPI copy on the minichromosome are viable on glucose media after counterselection. Human TPI, TPI_Ile170Val and TPI_Ile170Thr complemented for a loss of the TPI plasmid, but TPI_Lys13Arg did not. (b) TPI activity in yeast whole-cell extracts. Substrate conversion rates as normalized to total protein content. TPI_Ile170Val and TPI_Ile170Thr have lower activity than wild-type TPI. (c) Increased expression levels of TPI_Ile170Val and TPI_Ile170Thr in yeast as revealed by immunoblotting of whole-cell extracts using a TPI-specific antibody [35]. The amount loaded onto the SDS-PAGE gel was normalized to total protein, comparable loading was evaluated by Ponceau Red staining of the blotting membrane.