Figure 2. The serine hydrolase activity of LDAH is required for LasA toxicity.

(a) LDAH contains an α/β hydrolase domain (red) with a typical catalytic triad: S139 within a GxSxG motif, D271 and H300 (circled). A hydrophobic hairpin motif that mediates localization to lipid droplets (colored yellow) is present as an insertion in the α/β fold. Numbers refer to amino acid positions. (b) Abundance of LDAH protein in WT cells, LDAH^−/− cells (hereafter called LDAH^null) and LDAH^null cells re-expressing wild-type LDAH (LDAH^WT) or a mutant (LDAH^S139C) in which the catalytic triad serine (shown in A) was changed to cysteine. LDAH^WT and LDAH^S139C were expressed either as untagged proteins or fused to GFP (uncropped immunoblots are in Supplementary Fig. 10). Immunoblot was repeated three times with similar results. (c) LasA sensitivity of the indicated cell lines was assessed using an MTT assay. IC₅₀ values are as follows: 21 ± 1 nM for WT, 505 ± 22 nM for LDAH^null, 56 ± 3 nM for LDAH^null;LDAH^WT, 60 ± 3 nM for LDAH^null;LDAH^WT-GFP, 582 ± 25 nM for LDAH^null;LDAH^S139C and 599 ± 24 nM for LDAH^null; hLDAH^S139C-GFP. Circles denote the mean value (+/- S.D. from n=3 independent samples).