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The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, Saxena S, Gietz RD, Greenberg AH - J. Exp. Med. (1997)

Bottom Line: Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria.After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death.In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

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Subcellular localization of Nip3 and Nip3163. Rat-1 cells were transfected with HA-Nip3 and stained with anti-HA antibody using FITC (A)  and the mitochondrial protein marker anti-HSP60 antibody using Cy3 (B). The stained images were combined to compare the staining pattern of both  proteins (C) and their coincidence is indicated by the conversion of green FITC and red Cy3 stain to yellow thoughout the cell cytoplasm. HA-Nip3163  was expressed in Rat-1 cells then stained with anti-HA antibody (D), or anti-HSP60 antibody (E) and shown as a combined image using antibodies (F),  as described above.
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Figure 3: Subcellular localization of Nip3 and Nip3163. Rat-1 cells were transfected with HA-Nip3 and stained with anti-HA antibody using FITC (A) and the mitochondrial protein marker anti-HSP60 antibody using Cy3 (B). The stained images were combined to compare the staining pattern of both proteins (C) and their coincidence is indicated by the conversion of green FITC and red Cy3 stain to yellow thoughout the cell cytoplasm. HA-Nip3163 was expressed in Rat-1 cells then stained with anti-HA antibody (D), or anti-HSP60 antibody (E) and shown as a combined image using antibodies (F), as described above.

Mentions: Boyd et al. (21) reported that HA-Nip3 localizes in discrete cytoplasmic patches which are typical of a mitochondrial distribution. To confirm the mitochondrial localization of Nip3 and to test the hypothesis that it depends on the putative transmembrane domain, we transfected Rat-1 cells with HA-Nip3 or HA-Nip3163 then stained with FITC-conjugated anti-HA antibody. Cells were simultaneously treated with anti-HSP60 antibody which primarily stains mitochondria (15) and Cy3-conjugated second antibody. Both the green Nip3 and red HSP60 fluorescence appeared in a similar punctate pattern, and when the images were combined, a uniform yellow staining pattern indicated virtually complete coincidence of the two stains (Fig. 3, A–C). In contrast, HA-Nip3163 stained diffusely throughout the cytoplasm rather than the punctate distribution of the intact Nip3, however, a small amount of HA staining appeared to colocalize with HSP60 by double fluorescence (Fig. 3, D–F).


The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, Saxena S, Gietz RD, Greenberg AH - J. Exp. Med. (1997)

Subcellular localization of Nip3 and Nip3163. Rat-1 cells were transfected with HA-Nip3 and stained with anti-HA antibody using FITC (A)  and the mitochondrial protein marker anti-HSP60 antibody using Cy3 (B). The stained images were combined to compare the staining pattern of both  proteins (C) and their coincidence is indicated by the conversion of green FITC and red Cy3 stain to yellow thoughout the cell cytoplasm. HA-Nip3163  was expressed in Rat-1 cells then stained with anti-HA antibody (D), or anti-HSP60 antibody (E) and shown as a combined image using antibodies (F),  as described above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199165&req=5

Figure 3: Subcellular localization of Nip3 and Nip3163. Rat-1 cells were transfected with HA-Nip3 and stained with anti-HA antibody using FITC (A) and the mitochondrial protein marker anti-HSP60 antibody using Cy3 (B). The stained images were combined to compare the staining pattern of both proteins (C) and their coincidence is indicated by the conversion of green FITC and red Cy3 stain to yellow thoughout the cell cytoplasm. HA-Nip3163 was expressed in Rat-1 cells then stained with anti-HA antibody (D), or anti-HSP60 antibody (E) and shown as a combined image using antibodies (F), as described above.
Mentions: Boyd et al. (21) reported that HA-Nip3 localizes in discrete cytoplasmic patches which are typical of a mitochondrial distribution. To confirm the mitochondrial localization of Nip3 and to test the hypothesis that it depends on the putative transmembrane domain, we transfected Rat-1 cells with HA-Nip3 or HA-Nip3163 then stained with FITC-conjugated anti-HA antibody. Cells were simultaneously treated with anti-HSP60 antibody which primarily stains mitochondria (15) and Cy3-conjugated second antibody. Both the green Nip3 and red HSP60 fluorescence appeared in a similar punctate pattern, and when the images were combined, a uniform yellow staining pattern indicated virtually complete coincidence of the two stains (Fig. 3, A–C). In contrast, HA-Nip3163 stained diffusely throughout the cytoplasm rather than the punctate distribution of the intact Nip3, however, a small amount of HA staining appeared to colocalize with HSP60 by double fluorescence (Fig. 3, D–F).

Bottom Line: Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria.After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death.In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

Show MeSH
Related in: MedlinePlus