Figure 4.

ERH1 Encodes an IPCS.

(A) Complementation of the yeast aur1 mutation by ERH1. ERH1 was cloned in the pADH-LEU2 plasmid. The parental and the recombinant plasmids were introduced into three yeast strains lacking AUR1p alone (aur1Δ), AUR1p and ceramide-associated fatty acyl chain C2-hydroxylase (aur1Δ/scs7Δ), or AUR1p along with sphinganine hydroxylase (aur1Δ/sur2Δ). The respective yeast cells were grown on SD medium (−Leu) containing FOA.

(B) Yeast cells expressing ERH1 possess IPCS activities. Microsomes were prepared from aur1Δ yeast cells harboring pAUR1 or pERH1 and assayed for IPCS activities using the indicated ceramide substrates. Note that IPCS activities are reflected by the production of IPC (bottom bands) from the ceramide substrate (strong top bands). Cer, ceramide; Sph, sphingosine.

(C) In vitro assays for plant IPCS activity. Leaf lysates were prepared from 6-week-old plants and subjected to IPCS assays using dodecanoyl-NBD-d-erythro-DHS as a substrate. IPCS activity was measured using relative fluorescence units. Data are means ± se (P < 0.015 based on Student's t test of the values of the two genotypes; n = 4).

(D) erh1 results in ceramide accumulation in plants expressing RPW8. Sphingolipids were extracted from leaves of 8-week-old plants of the indicated genotypes and measured essentially as described previously (Markham and Jaworski, 2007). Values are means ± se (n = 5). Asterisks indicate significance at P < 0.01 compared with S5 based on Student's t test. dw, dry weight.