The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.
Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.
Borner
C
C
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Martinou
I
I
Mattmann
C
C
Irmler
M
M
Schaerer
E
E
Martinou
J C
JC
Tschopp
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002470:Cell Survival; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D006801:Humans; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D011505:Protein-Tyrosine Kinases