HUMAN PRO-TUMOR NECROSIS FACTOR - MOLECULAR DETERMINANTS OF MEMBRANE TRANSLOCATION, SORTING, AND MATURATION

Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane p rotein with a highly conserved 76-residue leader sequence. We have ana lyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplas mic, transmembrane, and linking domains. Cytoplasmic deletions include d the Arg doublet at -49 and -48 and/or the Lys doublet at -58 and -57 ; additional mutants included deletion of residues -73 to -55 and -73 to -55, -49, and -48. The transmembrane and linking domain mutants inc luded deletions in the -42 to -35 region, combined with the deletion o f residues -32 to -1. Two hybrid mutants combined the cytoplasmic dele tions with the deletion of residues -32 to -1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues -36, -35, -32 to -1, a lthough the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues -38 to -35 and -32 to -1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent e xpression and maturation of both the cytoplasmic and hybrid mutants. T hus, proper expression and maturation of human pro-TNF can be accompli shed with as few as similar to 12 of the 26 residues of the native tra nsmembrane domain and with a net negative charge in the cytoplasmic do main flanking the transmembrane region.