Figure S4.

Additional data supporting that SRD5A3 and Dfg10 convert polyprenal to dolichal, related to Figure 4

(A) Western blot analysis of protein extract from samples used for the preparation of membrane extracts showing expression of SRD5A3-N-His in WT HEK293T cells, detected by His immunoblotting. Overexpression was achieved using pcDNA3.1(+)-N-6His plasmid vector containing H. sapiens SRD5A3 cDNA with an N-terminal 6 x His-tag.

(B) Western blot analysis of protein extract from samples used for the preparation of membrane extracts showing expression of dfg10-N-His in WT HEK293T cells, detected by His immunoblotting. Overexpression was achieved using pcDNA3.1(+)-N-6His plasmid vector containing S. cerevisiae dfg10 cDNA appended to an N-terminal 6 x His-tag.

(C) Polyprenal reductase activity of SRD5A3 is strictly NADPH dependent. NADPH/NADH-dependence of SRD5A3-catalyzed polyprenal reductase activity in SRD5A3-overexpressing HEK293T membrane extracts in the presence of 0.3 mg/mL of membrane protein extract. 5 μg/mL polyprenal was used in the presence of 0, 5, 20, 100, 1000, or 5000 μmol/L of NADPH or NADH, 2h, 37°C. Data are TIC-normalized AUC (mean ± SEM, n = 3).

(D) Representative chromatograms, complementary to Figure 4A and (E), showing that significant dolichal-18 and dolichol-18 are only formed from polyprenal (5 μg/mL) in the presence of NADPH (5 mmol/L), but not NADH (5 mmol/L), after incubation for 2h at 37°C. PAL = polyprenal, POL = polyprenol, DAL = dolichal and DOL = dolichol.

(E and F) SRD5A3-dependent polyprenal and polyprenol reductase activity was assessed in membrane extracts from HEK293T cells (E) or DHRSX KO HAP1 cells (F) overexpressing SRD5A3 or an empty vector. Measurement of polyprenol-18, polyprenal-18, dolichol-18M + 2, and dolichal-18 from 0.3 mg/mL control or SRD5A3-overexpressing cells after incubation in the presence of polyprenal or polyprenol (5 μg/mL) and NADPH or NADH (5 mmol/L), 2h, 37°C. Data are TIC-normalized AUC (mean ± SEM, n = 3). Several metabolites are already shown in Figures 4A and 4B.

(G) Dfg10-dependent polyprenal reductase and polyprenol reductase activity was assessed in membrane-extract from HEK293T cells overexpressing S. cerevisiae dfg10. Measurement of polyprenol-18, polyprenal-18, dolichol-18M + 2, and dolichal-18 from 0.3 mg/mL control or Dfg10-overexpressing HAP1 cells after incubation in the presence of polyprenal or polyprenol (5 μg/mL) and NADPH or NADH (5 mmol/L), 2h, 37°C. Data are TIC-normalized AUC (mean ± SEM, n = 3). Several metabolites are already shown in Figures 4E and 4F.