Transmembrane TNF (pro-TNF) is palmitoylated

Human transmembrane tumor necrosis factor (pro-TNF) was examined for protei n acylation, The cDNA encoding pro-TNF was expressed in both COS-I cells an d Sf9 cells and metabolic labeling with [H-3]myristic or [H-3]palmitic acid was attempted, The 17 kDa mature TNF secreted from the transfected cells w as not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [H -3]palmitic acid. The [H-3]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to p almitoylation of a cysteine residue via a thioester bond. Site-directed mut agenesis of the two cysteine residues residing in the leader sequence of pr o-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains o f pro-TNF. Thus, pro-TNF interacts with the plasma membrane via both its pr oteinaceous transmembrane domain and a lipid anchor, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rig hts reserved.