Transmembrane TNF (pro-TNF) is palmitoylated.
Human transmembrane tumor necrosis factor (pro-TNF) was examined for protein acylation. The cDNA encoding pro-TNF was expressed in both COS-1 cells and Sf9 cells and metabolic labeling with [(3)H]myristic or [(3)H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [(3)H]palmitic acid. The [(3)H]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site-directed mutagenesis of the two cysteine residues residing in the leader sequence of pro-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains of pro-TNF. Thus, pro-TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.
Utsumi
T
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. utsumi@agr.yamaguchi-u.ac.jp
Takeshige
T
T
Tanaka
K
K
Takami
K
K
Kira
Y
Y
Klostergaard
J
J
Ishisaka
R
R
D000215:Acylation; D000818:Animals; D019556:COS Cells; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism*; D007313:Insecta; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005229:Fatty Acids, Monounsaturated; D014409:Tumor Necrosis Factor-alpha; C008757:palmitoleic acid; D003545:Cysteine