Figure 4.

SRD5A3 and its yeast orthologue Dfg10 produce dolichal from polyprenal (reaction 2) but have undetectable activity on polyprenol

(A and B) SRD5A3 shows activity on polyprenal but not on polyprenol. Formation of dolichal from polyprenal (reaction 2 of revised model of dolichol synthesis) (A) and formation of dolichol from polyprenol (previously accepted function of SRD5A3 and Dfg10) (B) were assessed using membrane preparations from HEK293T cells overexpressing human SRD5A3 or an empty vector control. Polyisoprenoids were quantified after 2 h incubation at 37°C with polyprenal (A) or polyprenol (B) at 5 μg/mL and NADPH or NADH at 5 mmol/L. Data represent TIC-normalized AUC (means ± SEM, n = 3). See also Figures S4D, S4E, and S4F.

(C) Activity of SRD5A3 determined by measuring dolichal formation after incubation of the indicated concentrations of polyprenal with 1 mmol/L NADPH and 1 μg/mL membrane preparations from DHRSX KO HAP1 cells overexpressing SRD5A3 for 30 min at 37°C (upper) or after an identical incubation of 4 μM of polyprenal with the indicated concentrations of NADPH (lower). Values were calculated based on the formation of dolichal-18 (means ± SEM; n = 3).

(D) Isoprenoid species in WT (BY4741), dfg10 KO, and dfg10 KO + dfg10 S cerevisiae. Data are TIC-normalized AUC (means ± SEM; n = 3; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.01; ^∗∗∗∗p < 0.0001). Species with 16 isoprenyl units are shown. See also Table S2.

(E and F) The SRD5A3 yeast orthologue Dfg10 also shows activity on polyprenal but not on polyprenol. Formation of dolichal from polyprenal (reaction 2 of revised model of dolichol synthesis) (E), and formation of dolichol from polyprenol (previously accepted function Dfg10) (F) are presented as described in (A), but using extracts from HEK293T cells overexpressing Dfg10. See also Figure S4G.