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p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA, Shore GC - J. Cell Biol. (1997)

Bottom Line: Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so.The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells.Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

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Insertion of p28 into ER microsomes. Pre–β-lactamase  (lanes 1–4) and p28 (lanes 5–8) mRNA was translated in a rabbit  reticulocyte lysate system in the presence of [35S]methionine, in  the presence (lanes 2–4 and 6–8) or absence (lanes 1 and 5) of ribosome-stripped canine pancreas microsomes (Walter and Blobel, 1983). At the end of the reaction, microsomes were recovered and analyzed by SDS-PAGE and fluorography either  directly (lanes 2 and 6) or after isolation of alkali-insoluble  (NaCO3, pH 11.5) product (lanes 3 and 7; Nguyen et al., 1993), or  after treatment with proteinase K (lanes 4 and 8; McBride et al.,  1992). The positions of p28, pre–β-lactamase (pre-β-L), and processed β-lactamase (β-L) are indicated, as is the gel front. c,  marker translation product. The schematic shown below the fluorogram depicts the deduced topology of p28 in the ER membrane  (see text).
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Figure 3: Insertion of p28 into ER microsomes. Pre–β-lactamase (lanes 1–4) and p28 (lanes 5–8) mRNA was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, in the presence (lanes 2–4 and 6–8) or absence (lanes 1 and 5) of ribosome-stripped canine pancreas microsomes (Walter and Blobel, 1983). At the end of the reaction, microsomes were recovered and analyzed by SDS-PAGE and fluorography either directly (lanes 2 and 6) or after isolation of alkali-insoluble (NaCO3, pH 11.5) product (lanes 3 and 7; Nguyen et al., 1993), or after treatment with proteinase K (lanes 4 and 8; McBride et al., 1992). The positions of p28, pre–β-lactamase (pre-β-L), and processed β-lactamase (β-L) are indicated, as is the gel front. c, marker translation product. The schematic shown below the fluorogram depicts the deduced topology of p28 in the ER membrane (see text).

Mentions: As shown in Fig. 3, p28 was efficiently inserted cotranslationally into dog pancreas microsomes. In contrast to β-lactamase, which was translocated across the ER membrane and deposited in the lumen as a soluble protein, p28 was recovered as an integral protein after release from the ribosome. Whereas the processed form of β-lactamase was protected from external protease (Fig. 3, lane 4) and liberated from microsomes by alkaline extraction (Fig. 3, lane 3), p28 was resistant to alkaline extraction (Fig. 3, lane 7), and exhibited sensitivity to external protease (Fig. 3, lane 8), resulting in the generation of proteolytic fragments that would be expected for a multispanning integral protein with an exposed cytosolic domain. Unlike β-lactamase, whose NH2-terminal signal sequence was removed during translocation (Fig. 3, compare lanes 1 and 4), processing of p28 was not observed (Fig. 3, compare lanes 5 and 6), suggesting that insertion into the microsomal membrane is initiated by an uncleaved signal anchor. Though not studied in detail, the observed properties of p28 (Fig. 3), together with predictions for the orientation of transmembrane segments in the ER based on charge difference rules (von Heijne, 1986; Hartmann et al., 1989), suggest a topology for p28 in the ER membrane in which the NH2 terminus of this triple-spanning polypeptide faces the lumen, leaving an ∼13-kD COOH-terminal fragment containing predicted caspase cleavage sites, leucine zipper/death effector-like domain, and ER retention motif exposed to the cytosol (see Fig. 3). Biochemical fractionation and cryoimmunocytochemical electron microscopy confirmed that p28 is predominantly located in the ER in rat hepatocytes (not shown).


p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA, Shore GC - J. Cell Biol. (1997)

Insertion of p28 into ER microsomes. Pre–β-lactamase  (lanes 1–4) and p28 (lanes 5–8) mRNA was translated in a rabbit  reticulocyte lysate system in the presence of [35S]methionine, in  the presence (lanes 2–4 and 6–8) or absence (lanes 1 and 5) of ribosome-stripped canine pancreas microsomes (Walter and Blobel, 1983). At the end of the reaction, microsomes were recovered and analyzed by SDS-PAGE and fluorography either  directly (lanes 2 and 6) or after isolation of alkali-insoluble  (NaCO3, pH 11.5) product (lanes 3 and 7; Nguyen et al., 1993), or  after treatment with proteinase K (lanes 4 and 8; McBride et al.,  1992). The positions of p28, pre–β-lactamase (pre-β-L), and processed β-lactamase (β-L) are indicated, as is the gel front. c,  marker translation product. The schematic shown below the fluorogram depicts the deduced topology of p28 in the ER membrane  (see text).
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Figure 3: Insertion of p28 into ER microsomes. Pre–β-lactamase (lanes 1–4) and p28 (lanes 5–8) mRNA was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, in the presence (lanes 2–4 and 6–8) or absence (lanes 1 and 5) of ribosome-stripped canine pancreas microsomes (Walter and Blobel, 1983). At the end of the reaction, microsomes were recovered and analyzed by SDS-PAGE and fluorography either directly (lanes 2 and 6) or after isolation of alkali-insoluble (NaCO3, pH 11.5) product (lanes 3 and 7; Nguyen et al., 1993), or after treatment with proteinase K (lanes 4 and 8; McBride et al., 1992). The positions of p28, pre–β-lactamase (pre-β-L), and processed β-lactamase (β-L) are indicated, as is the gel front. c, marker translation product. The schematic shown below the fluorogram depicts the deduced topology of p28 in the ER membrane (see text).
Mentions: As shown in Fig. 3, p28 was efficiently inserted cotranslationally into dog pancreas microsomes. In contrast to β-lactamase, which was translocated across the ER membrane and deposited in the lumen as a soluble protein, p28 was recovered as an integral protein after release from the ribosome. Whereas the processed form of β-lactamase was protected from external protease (Fig. 3, lane 4) and liberated from microsomes by alkaline extraction (Fig. 3, lane 3), p28 was resistant to alkaline extraction (Fig. 3, lane 7), and exhibited sensitivity to external protease (Fig. 3, lane 8), resulting in the generation of proteolytic fragments that would be expected for a multispanning integral protein with an exposed cytosolic domain. Unlike β-lactamase, whose NH2-terminal signal sequence was removed during translocation (Fig. 3, compare lanes 1 and 4), processing of p28 was not observed (Fig. 3, compare lanes 5 and 6), suggesting that insertion into the microsomal membrane is initiated by an uncleaved signal anchor. Though not studied in detail, the observed properties of p28 (Fig. 3), together with predictions for the orientation of transmembrane segments in the ER based on charge difference rules (von Heijne, 1986; Hartmann et al., 1989), suggest a topology for p28 in the ER membrane in which the NH2 terminus of this triple-spanning polypeptide faces the lumen, leaving an ∼13-kD COOH-terminal fragment containing predicted caspase cleavage sites, leucine zipper/death effector-like domain, and ER retention motif exposed to the cytosol (see Fig. 3). Biochemical fractionation and cryoimmunocytochemical electron microscopy confirmed that p28 is predominantly located in the ER in rat hepatocytes (not shown).

Bottom Line: Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so.The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells.Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

Show MeSH
Related in: MedlinePlus