Fig. 4.

Functional analysis of CAB1 variants. a Activity of PanK variants constructed by site-directed mutagenesis. The coding region of wild-type CAB1 was mutagenized at the positions indicated and gene variants were inserted into a multi-copy expression vector containing the MET25 promoter together with the HA epitope. The resulting plasmids pSBS5 (CAB1 wild-type), pEB5 (CAB1 Y326A F330A), pEB6 (cab1 I234E), pEB8 (CAB1 W331L), pEB22 (CAB1 N155V), pEB23 (cab1 S158V), pEB25 (cab1 R173A), pEB26 (CAB1 A233E), and pEB27 (CAB1 W331R) were transformed into strain JS91.14–24 (cab1^ts). Transformants were cultivated at 30 °C until the mid-log growth phase. Pantothenate kinase (PanK) activity in protein extracts prepared from transformants was assayed at 37 °C. For each assay, 75 μg of total protein was used. PanK activities are given in cpm 1-¹⁴C-phosphopantothenate formed per μg protein. Standard deviations are indicated by error bars. b Stable expression of PanK variants was investigated by Western blot analysis of protein extracts prepared from transformants using anti-HA antibodies