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 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 approximately 12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.
Utsumi
T
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Akimaru
K
K
Kawabata
Z
Z
Levitan
A
A
Tokunaga
T
T
Tang
P
P
Ide
A
A
Hung
M C
MC
Klostergaard
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004285:Dogs; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D006801:Humans; D007477:Ions; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D007477:Ions; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha