Effects of truncation of human pro-tumor necrosis factor transmembrane domain on cellular targeting.
Human tumor necrosis factor is initially synthesized as a transmembrane prohormone anchored by a hydrophobic region of the leader sequence. This hydrophobic domain has been previously localized to extend from Leu-46 to Ile-21 based on hydropathy calculations. To functionally determine the nature of this domain, we have generated a series of pro-TNF mutant cDNAs in which either half or both halves of this encoding domain is deleted. These cDNAs were analyzed both by the ability of their mRNAs to direct translation in a microsomal system and by cellular localization of their encoded TNFs following transfection of NIH/3T3 cells. We determined that the mutant protein with deletion of the periluminal region of the transmembrane domain (Thr-32 to Ile-21) was translocated into microsomes and localized on the inner surface of the microsomal membrane in a fashion identical to that of the parental TNF. In contrast, the mutants with deletion of either the pericytoplasmic aspect (Leu-46 to Gly-34) or of virtually the entire transmembrane domain were not localized in the microsomes. Transfection experiments indicated that only the cDNAs whose peptide products were translocated across microsomal membranes gave rise to transmembrane prohormones and matured TNFs. Thus, the functions of membrane targeting and orientation prior to proteolytic processing can be fulfilled by the sequence Leu-46 to Ala-33 of the transmembrane domain, but not by the sequence Ala-33 to Ile-21.
Utsumi
T
T
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Levitan
A
A
Hung
M C
MC
Klostergaard
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D014409:Tumor Necrosis Factor-alpha; C061153:tumor necrosis factor precursor