Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included 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 included deletions in the ؊42 to ؊35 region, combined with the deletion of residues ؊32 to ؊1. Two hybrid mutants combined the cytoplasmic deletions 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, although 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 expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as ϳ12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.

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