Localization and lipidation of LtgA and LtgD. (A) Strains containing C-terminal 3XFLAG at the native site of ltgA or ltgD were fractionated into outer membrane (OM), total membrane (TM), and soluble (SOL) fractions using ultracentrifugation. Anti-FLAG antibodies were used to determine the subcellular localization of LtgA and LtgD, which localized to the same fractions as the OM control, PilQ. The strains used had a chromosomal cat gene to identify the cytoplasmic fraction and the presence of SecY was used as an inner membrane control. (B) The incorporation of the lipid [3H]-palmitate into recombinant LTs producing wild-type (WT), cysteine to alanine lipidation mutations (LM), or proteins lacking signal sequences (SM). The (*) indicates an active site mutant was used.