A part of the transmembrane domain of pro-TNF can function as a cleavable signal sequence that generates a biologically active secretory form of TNF

To determine the minimum requirement in the 76-residue leader sequence of p ro-tumor necrosis factor (TNF) for membrane translocation across the endopl asmic reticulum (ER) and for the maturation of pro-TNF, we constructed pro- TNF mutants in which a part of the transmembrane domain of pro-TNF was dire ctly linked to the N-terminus of the mature domain, and evaluated their tra nslocational behavior across the ER-membrane and their secretion from the t ransfected cells, The in vitro translation/translocation assay involving a canine pancreatic microsomal membrane system including a mutant, Delta-75-4 7 7, -32-1, revealed that the N-terminal half of the transmembrane domain o f pro-TNF consisting of 14 residues functioned as a cleavable signal sequen ce; it generated a cleaved form of TNF having a molecular mass similar to t hat of mature TNF, Analysis of the cleavage site by site-directed mutagenes is indicated that the site was inside the leader sequence of this mutant. W hen the mutant, Delta-75-47, -32-1, was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further delet ion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the mem brane translocation of the mature domain of TNF, Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of a biolo gically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment. In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular proc essing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and thus enables the leader se quence to function as a type II signal-anchor sequence that generates a tra nsmembrane form of TNF.