Figure 3. Six alanine mutations affecting the regulation of PSS 1.

(A) CHO-K1 cells were transiently transfected with 6 μg of each of the control empty plasmid (vector), and plasmids that, respectively, encoded wild-type (wt) or R95A, H97A, C189A, R262A, Q266A and R336A mutant PSS 1. The transfectants were metabolically labelled with [¹⁴C]serine for 3 h at 37 °C in the growth medium supplemented with (hatched bars) or without (filled bars) 100 μM PtdSer, and then the radioactivity of cellular PtdSer was determined. Three independent experiments gave similar results; the values shown are for one of three independent experiments and are the averages of duplicate determinations with a variation of <7% between duplicates. (B and C) Samples (0.25 μg) of each of the same plasmids as those used in the experiments described in (A) were transfected into CHO-K1 cells. (B) Cell lysates of the transfectants were assayed for serine base-exchange activity in the presence (hatched bars) or absence (filled bars) of 100 μM PtdSer. Two independent experiments gave similar results; the values shown are for one of the two independent experiments, and are the averages of duplicate determinations with a variation of <11% between duplicates. (C) Cell lysates (10 μg of protein) of the transfectants were separated by SDS/PAGE, and then analysed by Western blotting with an anti-PSS 1 N-terminal peptide antibody.