FIGURE 3.

Purified Lnt is functional in vitro. A, Lnt N-acylates apo-Lpp in vitro. Purified Lnt was incubated with detergent-solubilized membrane vesicles derived from Lnt-depleted cells containing apo-Lpp as substrate and phospholipids (PL) as acyl donors. Samples were taken at different time points and analyzed by Tricine-SDS-PAGE and immunoblotting using anti-Lpp antibodies. Asterisks indicate Lpp covalently cross-linked to peptidoglycan fragments. B, Lnt-dependent [³H]palmitoyl incorporation in FSL-1. The diacylglyceryl-lipopeptide FSL-1 was mixed with phospholipid vesicles isolated from [³H]palmitate-labeled cells. Following the addition of Lnt (○) or inactive Lnt(K335A) (△), the reaction mixture was incubated at 37 °C, and samples were removed at time intervals. The peptide was purified via streptavidin-biotin affinity, and [³H]palmitate incorporation was determined by scintillation counting. The amount of [³H]palmitate incorporated into the peptide is expressed as a percentage of the total amount of radioactivity in the reaction mixture from three independent experiments.