The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis
CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-α nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-α nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. Chloride intracellular channel (CLIC) 1The abbreviations used are: CLICchloride intracellular channelERendoplasmic reticulumTNFtumor necrosis factorNLSnuclear localization signalPBSphosphate-buffered salineORFopen reading frameCMVcytomegalovirusHAhemagglutininFACSfluorescence-activated cell sortingPIpropidium iodideTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingGFPgreen fluorescent proteinNTFnuclear transport factorTMtransmembraneAdadenovirusApafApoptotic protease activating factorEMelectron microscopy.1The abbreviations used are: CLICchloride intracellular channelERendoplasmic reticulumTNFtumor necrosis factorNLSnuclear localization signalPBSphosphate-buffered salineORFopen reading frameCMVcytomegalovirusHAhemagglutininFACSfluorescence-activated cell sortingPIpropidium iodideTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingGFPgreen fluorescent proteinNTFnuclear transport factorTMtransmembraneAdadenovirusApafApoptotic protease activating factorEMelectron microscopy. is a recently identified family of proteins with seven members (p64, CLIC1–5, and parchorin) that are localized in various cellular compartments and expressed in multiple tissue types. The cellular functions of CLIC family members may involve organellar volume regulation and ionic homeostasis and regulation of electro-neutrality (1Jentsch T.J. Nature. 2002; 415: 276-277Crossref PubMed Scopus (43) Google Scholar). Five of the CLIC proteins are similar in size and highly homologous to the C terminus (CLIC module) of p64 and parchorin, which in addition consist of distinct N-terminal domains (2Ashley R.H. Mol. Membr. Biol. 2003; 20: 1-11Crossref PubMed Scopus (104) Google Scholar). All members have one major hydrophobic stretch in the CLIC module that may be used to span the membrane, but some members are also found in a soluble form in the cytoplasm (3Qian Z. Okuhara D. Abe M.K. Rosner M.R. J. Biol. Chem. 1999; 274: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 4Edwards J.C. Am. J. Physiol. 1999; 276: F398-F408PubMed Google Scholar, 5Mizukawa Y. Nishizawa T. Nagao T. Kitamura K. Urushidani T. Am. J. Physiol. 2002; 282: C786-C795Crossref PubMed Scopus (24) Google Scholar, 6Tulk B.M. Kapadia S. Edwards J.C. Am. J. Physiol. 2002; 282: C1103-C1112Crossref PubMed Scopus (93) Google Scholar). Soluble CLIC proteins are structural homologues of soluble omega-class glutathione S-transferase family proteins, and they may behave as an “active” anion channel and/or channel regulator when “autoinserted” to form integral membrane proteins (7Harrop S.J. DeMaere M.Z. Fairlie W.D. Reztsova T. Valenzuela S.M. Mazzanti M. Tonini R. Qiu M.R. Jankova L. Warton K. Bauskin A.R. Wu W.M. Pankhurst S. Campbell T.J. Breit S.N. Curmi P.M. J. Biol. Chem. 2001; 276: 44993-45000Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). chloride intracellular channel endoplasmic reticulum tumor necrosis factor nuclear localization signal phosphate-buffered saline open reading frame cytomegalovirus hemagglutinin fluorescence-activated cell sorting propidium iodide terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling green fluorescent protein nuclear transport factor transmembrane adenovirus Apoptotic protease activating factor electron microscopy. chloride intracellular channel endoplasmic reticulum tumor necrosis factor nuclear localization signal phosphate-buffered saline open reading frame cytomegalovirus hemagglutinin fluorescence-activated cell sorting propidium iodide terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling green fluorescent protein nuclear transport factor transmembrane adenovirus Apoptotic protease activating factor electron microscopy. Mitochondrial CLIC (mtCLIC/CLIC4) was the first homologue of p64 identified, and its mRNA is ubiquitously expressed with the highest expression in lung, brain, liver, kidney, and skin (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). CLIC4 was described as a mitochondrial and cytoplasmic protein in mouse keratinocytes and localized to the inner mitochondrial membrane in human keratinocytes (9Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). Other reports indicate CLIC4 is also localized in the trans-Golgi network in pancreatic cells, endoplasmic reticulum (ER) in rat hippocampal HT-4 cells, and large dense core vesicles in neurosecretory cells (10Chuang J.Z. Milner T.A. Zhu M. Sung C.H. J. Neurosci. 1999; 19: 2919-2928Crossref PubMed Google Scholar, 11Duncan R.R. Westwood P.K. Boyd A. Ashley R.H. J. Biol. Chem. 1997; 272: 23880-23886Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 12Edwards J.C. Tulk B. Schlesinger P.H. J. Membr. Biol. 1998; 163: 119-127Crossref PubMed Scopus (54) Google Scholar). Similar to other family members (parchorin, p64, CLIC1, CLIC3), CLIC4 has a putative Cl--selective channel activity exhibiting a single channel conductance (2Ashley R.H. Mol. Membr. Biol. 2003; 20: 1-11Crossref PubMed Scopus (104) Google Scholar). CLIC4 homologues are expressed in many species with 85–95% identity at the amino acid level suggesting physiological functions are evolutionarily conserved (13Shorning B.Y. Wilson D.B. Meehan R.R. Ashley R.H. Dev. Genes Evol. 2003; 213: 514-518Crossref PubMed Scopus (13) Google Scholar). CLIC4 has also been associated with the actin cytoskeleton in membrane ruffles and lamellipodia and interacts dynamically with signaling proteins involved in cell membrane remodeling, including dynaminI, actin, tubulin, and 14-3-3ϵ. O. isoforms in neuronal cells (14Suginta W. Karoulias N. Aitken A. Ashley R.H. Biochem. J. 2001; 359: 55-64Crossref PubMed Scopus (90) Google Scholar). Recently, substantial up-regulation of CLIC4 was reported in transforming growth factor-β and serum-activated human breast fibroblasts, where up-regulation of CLIC4 was associated with transdifferentiation to myofibroblasts (15Ronnov-Jessen L. Villadsen R. Edwards J.C. Petersen O.W. Am. J. Pathol. 2002; 161: 471-480Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Our previous studies have shown that CLIC4 is a direct response gene for p53 transactivation and involved in TNF-α- and p53-mediated signaling pathways. CLIC4 overexpression induces apoptosis associated with loss of mitochondrial membrane potential, cytochrome c release, and caspase activation suggesting that apoptosis is mediated by mitochondrial dysfunction (9Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). During the course of our studies on CLIC4 and apoptosis, have that cytoplasmic CLIC4 to the nucleus in cells p53-mediated apoptosis. studies have that nuclear translocation of CLIC4 was detected in cells to various stress and apoptotic stress and is mediated by the nuclear localization signal and the nuclear import CLIC4 is to the apoptosis is and in the of caspase These results that CLIC4 participates in the apoptotic response in cellular from and as cells in with as described T. Weinberg W.C. Yuspa S.H. PubMed Scopus Google Scholar). The cell was from mouse keratinocytes and in the as keratinocytes. The human p53 cell A.C. S. K. Genes Dev. 1997; PubMed Scopus Google was in and addition of p53 treated with inducers and in and the treated cells at various for adenovirus was from mouse fibroblasts a of and and as described R. 1999; PubMed Scopus Google Scholar). All shown at terminus CLIC4 and by the as described (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The terminus CLIC4 are for CLIC4 and used in including and cells by cell and with and at for lysates by a membrane, and the protein expression was by some as described (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). cytochrome c and actin p53 Importin-α and cytochrome c used at by the and to and used for the cells with in and with a and at for and the was as the The cell of was at for with protein and with the protein with CLIC4 at These in and prior to CLIC4 and CLIC4 open reading frame was with and and The was to and the cytoplasm mitochondria and nucleus with the C terminus by the and by and confocal CLIC4 used as to CLIC4 adenoviral and used to the CLIC4 and to and from and by and from by and to the by the in the of Deletion of the and of the that are to various of mouse CLIC4 to deletion in the of the CLIC4 to and the and to and the and by and deletion by a and to and to and the and with the including and confocal microscopy. Deletion CLIC4 proteins in cells with or adenovirus and the proteins expressed at by the of CLIC4 from was with and of with the N terminus and was by and was to amino and in the to to the The was and The was as described by the and the CLIC4 was by and was used as a for by multiple or with null as a was used to cell as described (9Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). keratinocytes and cells or with CLIC4 by on a various or cells and in of and in the or of propidium iodide for the of cell as a of apoptotic cells from at and with an as described by the in for in and by by confocal cells in with propidium for with with as described by the cells in with with fluorescent and the confocal The cells from the confocal and by the of cells to the of used to the of the cells for in for and with the of the with on a with and the cells with The cells for with and a was as described by the cells with and by confocal microscopy. The of cells was the as described for the keratinocytes with CLIC4 at and to as described by the was by a on in for and with cells with for and and a and with treated in for with for with and for or in The cells with of for with and in the with for The in treated with nuclear for with and with for with fluorescent and cells with the confocal microscopy K. 1997; PubMed Scopus Google was from the cell in in for in in a of in of and in in and at for on and in a of at The with and for in of for at The as and in and The with an electron with a of CLIC4 in of CLIC4 is localized in the in mitochondria and is (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). CLIC4 intracellular localization was in cells, that are or on mouse keratinocytes. of CLIC4 from the cytoplasmic the nucleus was detected in of the cells for cell and and and for the nuclear translocation was for the translocation prior to of apoptotic that CLIC4 nuclear translocation may be a the cellular response to stress. used at the nuclear translocation the of confocal the level of mitochondrial CLIC4 to be CLIC4 is a and and the signal be with the addition of the confocal of CLIC4 is an and proteins also to the nucleus or translocation of CLIC4 from the to the nucleus cytoplasmic or nuclear-targeted localized in the intracellular and to the nucleus in response to or a of CLIC4 nuclear was detected by of keratinocytes with or the confocal microscopy a of CLIC4 in the as in the confocal be to mitochondrial of the or a the and cytoplasm in nuclear by microscopy. from a cell be the nuclear translocation is by N terminus transmembrane and C terminus nuclear localization signal of CLIC4 deletion proteins with C terminus with the The shown the C terminus N terminus and the N terminus keratinocytes with the deletion and they other or to for and CLIC4 localization was detected by with by confocal microscopy. the with N terminus amino acid at the to was used to by or at was detected by and confocal microscopy. nuclear is in the keratinocytes with deletion by or at and the apoptotic cells detected by confocal microscopy. The of apoptotic cells was by or cells from of confocal CLIC4 to the in p53-mediated human CLIC4 and CLIC4 is p53-mediated apoptosis in p53 cells (9Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). response to CLIC4 from the cytoplasm to the nucleus a when cells the apoptotic CLIC4 nuclear translocation is associated with apoptosis an as as from with agents. The response is also detected in human cells as as CLIC4 nuclear translocation also in p53 null cell and or that translocation is a p53 E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google and CLIC4 with the the of mouse keratinocytes with or an of CLIC4 with Ran, and transport factor is an of the nuclear import suggesting that CLIC4 is the nucleus by an nuclear transport the of CLIC4 with Ran, and cells and mouse a level of with and also be detected in cells, and the with Importin-α may be suggesting that nuclear may be to some in and cell a level of nuclear CLIC4 is in p53 cells analyses of CLIC4 with Ran, and in or keratinocytes the nuclear transport in CLIC4 to and microscopy was to the of CLIC4 in the nucleus in keratinocytes and keratinocytes treated with CLIC4 was localized to the nuclear membrane and in the in and treated in multiple that the of nuclear by in the cells compared with the cells, of CLIC4 nuclear the nuclear of the associated with the nuclear membrane, and in the indicate that CLIC4 participates in a in the other a chloride channel or a regulator on the nuclear CLIC4 localization to nuclear was also detected in cells that CLIC4 nuclear translocation is mediated by the nuclear transport The and N in of CLIC4 putative nuclear localization signal on the C terminus of CLIC4 (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google that the may be involved in the nuclear and proteins keratinocytes that treated with that the by a large nuclear translocation, whereas the N-terminal nuclear translocation at a level the for CLIC4 nuclear translocation, a of deletion was and cells treated with and Deletion the putative to the suggesting the is for CLIC4 nuclear with the but the N terminus to the nucleus in the of whereas the protein with and in the suggesting that the N terminus of the protein may be involved in nuclear transport the proteins, only the protein to the nucleus suggesting that the N terminus a in regulation of CLIC4 nuclear Other and domains in the cytoplasm that the N and C of CLIC4 are for the nuclear that the CLIC4 C terminus with the putative is for the nuclear the CLIC4 is a amino in the to the The CLIC4 to the nucleus or whereas the and the CLIC4 to the nucleus with or These results indicate that the is for CLIC4 nuclear deletion the N terminus and found in the nucleus prior to the nuclear localization of apoptosis. of the CLIC4 keratinocytes apoptosis by but expression of and deletion to apoptosis of the C terminus and also to apoptosis suggesting N and C of CLIC4 are to nuclear translocation and to apoptosis when the protein is CLIC4, but studies that up-regulation of CLIC4 induces apoptosis in cell (9Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). to apoptosis was to a and CLIC4 with a and The addition of the CLIC4 be detected as a in size microscopy of keratinocytes with proteins to the These used as to adenoviral of cells with the CLIC4 expression similar with and the CLIC4 expression a at The on and the size of and but the size of was suggesting that the mitochondrial signaling to CLIC4 was and in mitochondria as N. N. Biol. 2002; PubMed Scopus Google Scholar). of with and keratinocytes of apoptosis, including activation of of and up-regulation of keratinocytes with or at These in apoptotic detected at a when CLIC4 suggesting that CLIC4 to the nucleus or mitochondria accelerates apoptosis. of protein also cause used to the nucleus and of and proteins in keratinocytes apoptosis on suggesting the apoptosis and in protein of apoptotic organellar apoptotic was when CLIC4 keratinocytes by in for and the of and cells by detected by confocal microscopy and by and a and the of cytochrome c from the to the soluble in keratinocytes in mitochondrial membrane that the accelerates the apoptotic but a mitochondrial apoptotic is also associated with the nuclear apoptosis. The mitochondrial was associated with CLIC4 when keratinocytes and at (9Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google overexpression in the nucleus accelerates apoptosis and causes cytochrome c the CLIC4 used to and apoptotic cells by and confocal and by and The in cell by and the cell found in other when is compared with or by in the adenoviral cells to to and The used as the to cytochrome c and the of cytochrome c to cytoplasm was detected by the or was used as and the used as the of CLIC4 of apoptosis mediated by the CLIC4 the of a apoptotic Apoptotic protease activating factor null and cell with the CLIC4 and apoptosis was by a Apaf null cells of cells when compared with or whereas the apoptosis in and Apaf cells was similar and a similar of cells in and Apaf null cells and that cell a for cytochrome in and indicate be in CLIC4 to the nucleus in Apaf null and cells treated with and that CLIC4 signaling is in the of with the from other cell CLIC4 nuclear translocation in Apaf null and cells prior to apoptotic that nuclear apoptosis the keratinocytes with CLIC4 to by adenoviral overexpression the apoptosis by but to the apoptosis nuclear CLIC4 may multiple apoptotic pathways. The of CLIC4 nuclear translocation in response to a of that are by is to the cellular response to stress. translocation of CLIC4 is detected in response to and and with various and of translocation and prior to a or of or apoptosis. is by the translocation in keratinocytes treated with where translocation prior to a in CLIC4 an the of CLIC4 for and suggesting are involved in the cellular in to the and to the cytoplasm D. Dev. Biol. 1999; PubMed Scopus Google Scholar). The of CLIC4 with by may an form of protein including CLIC4 Importin-α of the and an nuclear import Biol. 2001; PubMed Scopus Google Scholar). CLIC4 may nuclear import W. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google an nuclear import to for the translocation of CLIC4 the translocation of to the nucleus with the import W. M. C. J. J. Cell. Biochem. 2001; 1-11Crossref PubMed Scopus Google Scholar). Importin-α and CLIC4 is also in cells, that a of CLIC4 may be to the protein of also to be with CLIC4 and a level of CLIC4 in the nucleus as by fluorescent signal with confocal microscopy and the of in the nucleus of keratinocytes detected by These results CLIC4 a physiological in the and may to the of cytoplasmic CLIC4 to the nucleus stress. is that the response of CLIC4 to various is in the by proteins J.M. S. 1998; PubMed Scopus Google and to mitochondria to cell CLIC4 has and interacts with a (14Suginta W. Karoulias N. Aitken A. Ashley R.H. Biochem. J. 2001; 359: 55-64Crossref PubMed Scopus (90) Google Scholar). that CLIC family members with the protein M. Edwards J.C. Urushidani T. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). p64 is a for the J.C. Kapadia S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (24) Google and associates with (3Qian Z. Okuhara D. Abe M.K. Rosner M.R. J. Biol. Chem. 1999; 274: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). is that alterations in or protein are involved in CLIC4 and studies The putative of CLIC4 to a for nuclear Deletion studies indicate that CLIC4 the but the N terminus are to to the nucleus apoptosis. the or CLIC4 the and in the cytoplasm in the of but only the in response to stress the of CLIC4 an an M.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and studies direct that is a that participates and the CLIC4 nuclear is with the that CLIC proteins may as an form in the cytoplasm and an form of that the is by the N terminus of the protein in the form in the cytoplasm (7Harrop S.J. DeMaere M.Z. Fairlie W.D. Reztsova T. Valenzuela S.M. Mazzanti M. Tonini R. Qiu M.R. Jankova L. Warton K. Bauskin A.R. Wu W.M. Pankhurst S. Campbell T.J. Breit S.N. Curmi P.M. J. Biol. 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Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). our have a cytoplasm and overexpression of cause apoptosis by CLIC4 in various the of CLIC4 in a for a protein of CLIC4 is to of an apoptotic the suggesting that nuclear translocation cellular stress to the apoptotic We an apoptotic response to nuclear-targeted or to deletion of CLIC4 that in the that nuclear CLIC4 has a addition to the of translocation with multiple stress of that nuclear translocation of CLIC4 has a in cellular apoptosis. CLIC4 causes cytochrome c from mitochondria of keratinocytes. nuclear of CLIC4 causes apoptosis in Apaf null a for the cytochrome of CLIC4 to the nucleus of Apaf null treated with and of cellular apoptosis of a activation has been reported in cell S.H. S. J. Biol. 2002; PubMed Scopus Google Scholar, L. J. D. K. S. Adams J.M. A. Nature. 2002; PubMed Scopus Google Scholar). previous studies indicate that by or in Apaf null is associated with mitochondrial including loss of mitochondrial membrane potential, but the to loss of has been K. M. K. J. Biochem. 2001; PubMed Scopus Google Scholar). translocation of CLIC4 may be one of the that is in the cytoplasm and the nucleus is a activity of Apoptotic nuclear a used to the of cell in multiple cell We now show that CLIC4 to the and its may in of and chloride that be involved in the apoptotic in the as activation of and A. Biol. PubMed Scopus Google Scholar, S. Biochem. 2002; PubMed Scopus Google Scholar, S. J. 1997; PubMed Scopus Google Scholar). Our studies CLIC4 localized to the nuclear membrane, and in studies have that CLIC4 translocation is associated with in nuclear chloride be a which CLIC4 apoptosis. was recently reported that CLIC4 are in mitochondria from cells, and mitochondria mitochondria T. L. A. S. T. N. C. M. J. 2003; PubMed Scopus Google Scholar). the that CLIC4 has studies indicate that CLIC4 participates in transdifferentiation of fibroblasts to myofibroblasts and cells to (15Ronnov-Jessen L. Villadsen R. Edwards J.C. Petersen O.W. Am. J. Pathol. 2002; 161: 471-480Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, A. M. K. T. T. M. Biochem. 2001; PubMed Scopus Google Scholar). These a for CLIC4 in cell signaling and be to a nuclear of channel translocation also be associated with of CLIC4 protein of CLIC4 in the cytoplasm is The of an channel protein in the nucleus and mitochondria that CLIC4 participates in the in major for the apoptotic response and have We and of the of for the cell of the and of the for Apaf null and cell of the of the for adenovirus and of the for We and for