Mitochondria Localization and Dimerization Are Required for CIDE-B to Induce Apoptosis

• DOI: 10.1074/jbc.c000207200
• Publication Date: 2002-07-26T15:04:49Z
• Authors: Zhengming Chen; Ke Guo; Shen Yon Toh; Zhihong Zhou; Peng Li
• Keywords: Fragmentation

Cell death-inducingDFF45-like effector (CIDE)-B is a member of the novel family apoptosis-inducing factors that share homology with N-terminal region DFF, DNA fragmentation factor. The molecular mechanism CIDE-B-induced apoptosis unclear. We have shown here CIDE-B protein localized in mitochondria and forms homodimers heterodimers other members. Serial deletion analyses suggest localization signal dimerization interface are overlapped to 30 amino acid residues at C-terminal CIDE-B. Mitochondria both required for apoptosis. Our study has thus revealed by formation high affinity homo- or heterodimeric complex. factor cell polymerase chain reaction green fluorescent phosphate-buffered saline Chinese hamster ovary major organelles respond death stimuli releasing such as cytochrome c altering cellular reduction-oxidation (redox) potential oxidative phosphorylation (1Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 2Li P. Jijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text PDF Scopus (6182) 3Susin S.A. Lorenzo H.K. Zamzami N. Marzo Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini Loeffler Larochette Goodlett Aebersokd R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref (3430) Scholar). A number pro-and anti-apoptotic proteins reside including various caspases (4Porter A.G. Trends Biol. 9: 394-401Abstract (87) Scholar), ced-4 ced-9 (5Chen F. Hersh B.M. Conradt B. Zhou Z. Riemer Gruenbaum Y. Horvitz H.R. 2000; 287: 1485-1489Crossref (193) Scholar) Bcl-2 (6Gross McDonnell Korsmeyer S.J. Genes Dev. 13: 1899-1911Crossref (3238) Nix (7Yasuda Theodorakis Subramanian T. Chinnadurai Chem. 273: 12415-12421Abstract (189) 8Chen Cizeau Velde C.V. Park J.H. Bozek Bolton Shi L. Dubik Greenberg A. 274: 7-10Abstract (237) important activity pro-apoptotic abrogates their activity. Many members can form either 9Ray Chen Vande V.C. Cizeay Gietz R.D. A.H. 275: 1439-1448Abstract (286) Scholar,10Gross Jockel Wei MC. EMBO 17: 3878-3885Crossref (964) Activation Bax appears induce subcellular translocation from cytosol well homodimerization (11Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. 94: 3668-3672Crossref (1023) Mutational conserved BH3 domain plays an role mediating heterodimerization Scholar).The (DFF)1 (12Enari Sakahira H. Okawa O. Iwamatsu Nagata 391: 43-50Crossref (2795) 13Liu Zou Slaughter C. (1997). 89: 175-184Abstract (1638) consists two subunits, nuclease (CAD/DFF40) its inhibitor (DFF45/ICAD). DFF45 chaperone function associating DFF40 (14McCarty J.S. Toh S.Y. Li Biochem. Biophys. Res. Commun. 264: 176-180Crossref (31) 15McCarty 181-185Crossref (35) effectors (CIDEs) was identified domains (16Inohara Koseki Wu Nunez 2526-2533Crossref (279) 17Inohara Death Differ. 6: 823-824Crossref (25) CIDE be divided into CIDE-N domain, which shares DFF40/45, CIDE-C within only Although this class DFF45/40 region, functions differ significantly. Unlike DFF45, over-expression CIDEs mammalian cells shows strong death-inducing (CIDE-C) being sufficient recently participated solving structure (or CIDE-N) (18Lugovskoy A.A. Chou J.J. McCarty Wagner 99: 747-755Abstract (95) structural suggested interact each low binding surface bipolar property consisting oppositely charged regions. This homophilic association strongly suggests weak interaction functioning regulatory Scholar).To gain insight induces apoptosis, we characterized localization. observed mitochondrially Systematic analysis showed responsible dimerization. data also Therefore, mitochondrial require function.RESULTS AND DISCUSSIONTo characterize CIDE-B, GFP coding fused ATG human transiently transfected COS-1 determine fusion full-length were present punctate structures cytoplasm. Co-staining these MitoTracker pattern similar GFP-CIDE-B, merging images almost completely overlapping patterns (Fig.1). Golgi endoplasmic reticulum-specific markers no staining (data not shown). specific due GFP, alone diffused distribution cytoplasm nucleus higher levels than Immunocytochemical HA- Flag-tagged expression show). our indicate mitochondria.The region(s) further defined generating containing truncated (Fig.2 A). (CIDE-BΔC) In contrast, (CIDE-BΔN) found spherically shaped granules predominantly (Fig. 1). end (CIDE-B-(148–219)) still mitochondria. However, 39 (CIDE-B-(118–180)) disrupted proteins, suggesting Surprisingly, (CIDE-B-(181–219)) expressed evenly throughout any organelle-like structure. regions 118 148 (CIDE-B-(118–148)) 180 (CIDE-B-(148–180)) did show typical either. To explore possibility disruption around may result abolishing signal, generated constructs span near 180. Indeed, residue 166–219 (CIDE-B-(166–219)) 148–195 (CIDE-B-(148–195)) all displayed minimal tested 166–195 (CIDE-B166–195, Fig. 1), indicating necessary target Computer it contains long stretch α-helical help insert membrane.Cell staurosporine etoposide GFP-CIDE-B nuclei shown), exerts By localizing mitochondria, directly Bcl-XL chelate thereby inducing It possible change membrane potential, induction production reactive oxygen species, release factors.Our previous biochemical acts through interaction, whereas synergistic requires contribution 18Lugovskoy contain multiple domains, co-expressed HA-tagged CIDE-A 293T cells. Flag-CIDE-B immunoprecipitated monoclonal antibodies against Flag epitope, co-precipitating detected HA antibodies. HA-CIDE-B co-precipitated but control 2 B), itself. cannot exclude order oligomers, assume will refer (HA-CIDE-BΔN) (HA-CIDE-BΔC) Flag-CIDE-B, mediates stable dimer formation. confirmed cotransfecting HA-CIDE-BΔN Flag-CIDE-BΔN Flag-CIDE-BΔC, respectively. co-immunoprecipitated Flag-CIDE-BΔC C). Reciprocal experiments using immunoprecipitate complex detecting product yielded same results Similar conducted between Flag-CIDE-A interacts D), mediated region. Consistent data, CIDE-BΔC/CIDE-BΔC C) detectable co-immunoprecipitation Scholar,18Lugovskoy delineate dimerization, cotransfected GFP-tagged antibody-immunoprecipitated products probed detect proteins. agreement above, CIDE-BΔN bound (Fig.3 A, lane 2). Deletion acids had little effect on homodimeric 148–219 3). abolished 4), CIDE-B/CIDE-B interaction. alone, middle (residue 148–180), 118–148) lanes 5-7). disrupt interface, ability 166–219, 148–195, co-immunoprecipitate flag-CIDE-B. 3 8-10). These A) Quantitative CIDE-B-(148–219) much (27- 20-fold amounts co-immunoprecipitated, respectively) compared CIDE-B-(166–195) (minimal B). upstream downstream Interestingly, coincides signal. Dimerization oligomerization) increase local concentration effectively death.Figure 3Serial interface. located C terminus (residues 166–195). CIDE-ΔN truncations co-transfected Flag-CIDE-B. (IP) antibodies.IB, immunoblot. B, relative truncations. intensity mutant measured densitometer scan (Bio-Rad) normalized deduce fold difference (CIDE-B166–195).View Large Image Figure ViewerDownload Hi-res image Download (PPT)As signals activating machinery critical many whether effect, level comparable (70 65%, respectively). effective (55%). (CIDE-B-(118–180)), neither nor dimerized itself, apoptotic (20%). Truncations (CIDE-B-(118–148), -(148–180), -(181–219)), localization, triggering deletions (CIDE-B-(166–219), -(148–195), -(166–195)) demonstrated slightly vector significantly lower CIDE-B-(148–219). differences levels, truncation mutants (Fig.4, inset). targeting very interesting identify (from 166 195) Single-point mutations abolish one activities would useful distinguishing roles.Figure 4Apoptosis induced hCIDE-B mutants. μg indicated plasmids 0.5 pCMV-β-galactosidase CHO Apoptotic quantitated, percentage calculated total β-galactosidase-positive error bars standard deviation determined least four independent measurements. inset Western blot antibody showing used assay.View (PPT) function. RESULTS death.As membrane. factors. death. As roles. grateful Lai Ping Yaw providing valuable reagents technical help. thank Dr. John comments manuscript.