The apoptosis mediator mDAP-3 is a novel member of a conserved family of mitochondrial proteins.
Programmed cell death is essential for organ development and regeneration. To identify molecules relevant for this process, full length cDNA cloning of a short, developmentally regulated murine cDNA fragment, MERM-3, was performed and showed a 1.7 kb mRNA encoding a 45 kDa protein with an ATP/GTP binding motive (P-loop). Sequence analysis revealed an 82% amino acid identity to the human death associated protein 3 (hDAP-3), a positive mediator of apoptosis. The full length sequence being the murine orthologue of hDAP-3 is therefore referred to as mDAP-3. In situ hybridization and northern blot analysis showed an abundant mRNA expression with a pronounced expression in highly proliferative epithelial compartments. For mDAP-3, cytochrome c release and induction of cell death could be demonstrated by overexpression of a mDAP-3/EGFP fusion protein. DAP-3 mediated apoptosis was shown to depend on a functional P-loop. Intracellular localization studies using the mDAP-3/EGFP fusion protein, cell fractionation and protease protection experiments localized mDAP-3 to the mitochondrial matrix. DAP-3, in contrast to cytochrome c, retained its mitochondrial localization during apoptosis induction. A mutant of a putative yeast orthologue of mDAP-3, YGL129c, here referred to as yDAP-3, has been shown to exhibit disrupted mitochondrial function. yDAP-3 deficient mutants could be shown to progressively loose mitochondrial DNA. Loss of mitochondrial DNA in yDAP-3 was partially prevented by transfection of the yDAP-3 deficient mutant with mDAP-3, indicating functional complementation by murine DAP-3 in the yeast system. These data identify mDAP-3 as one of the first proapoptotic factors in the mitochondrial matrix and provide evidence for a critical, evolutionary conserved role of members of the DAP-3 protein family for mitochondrial biogenesis.
Berger
T
T
Medizinische Poliklinik and Physiologische Chemie, Universität München, Germany. kretzler@medpoli.med.uni-muenchen.de
Brigl
M
M
Herrmann
J M
JM
Vielhauer
V
V
Luckow
B
B
Schlöndorff
D
D
Kretzler
M
M
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D017173:Caenorhabditis elegans / Q000737:chemistry; D002458:Cell Fractionation; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004272:DNA, Mitochondrial / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009928:Organ Specificity; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012269:Ribosomal Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C097041:DAP3 protein, human; D004272:DNA, Mitochondrial; C490762:Dap3 protein, mouse; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins