Limits...
Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane.

Kaufmann T, Schlipf S, Sanz J, Neubert K, Stein R, Borner C - J. Cell Biol. (2003)

Bottom Line: Bcl-2 lacks the signal and therefore localizes to several intracellular membranes.The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM.These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, D-79106 Freiburg, Germany.

ABSTRACT
It is assumed that the survival factors Bcl-2 and Bcl-x(L) are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-x(L) is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-x(L) requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.

Show MeSH

Related in: MedlinePlus

Bcl-xL and Bcl-xS specifically localize to the MOM, whereas Bcl-2 resides on several membranes. (A) Anti–Bcl-2 and anti–Bcl-x Western blots of subcellular fractions from parental HEK 293 cells (endo) or HEK293 cells transiently overexpressing Bcl-2, Bcl-xL, or FLAG–Bcl-xS (exo). The mitochondrial fraction was obtained from the 1.4 M band and the heavy microsomal fraction from the 1.0 M band of the sucrose gradient (see Materials and methods). Light microsomes are the pellet of the 100,000 g spin. Purity of the fractions was checked with anti-grp78/Bip and anti-KDEL (microsomes) and anti–COX-VIc (mitochondria) antibodies. TAP(I-VI)–EGFP contains the first six membrane-spanning regions of the antigen peptide transporter I (TAP I) fused to EGFP. This protein specifically spans the ER membrane and is detected by anti-GFP Western blotting after transient transfection. (B) Anti–Bcl-x Western blots of mitochondrial matrix, inner membrane (mb), and outer membrane fractions of rat liver, HEK293 cells, and HEK293 cells transiently overexpressing Bcl-xL (HEK293/Bcl-xL). (C) Anti–Bcl-2 and anti–Bcl-x Western blots of mitochondria or microsomes from parental HEK293 cells or HEK293 cells overexpressing Bcl-2 or Bcl-xL or FLAG–Bcl-xS, extracted directly with detergent (total) or first treated with sodium carbonate (pH 12, peripheral) and then extracted with detergent (integral). (D) Autoradiography of [35S]methionine-labeled, in vitro–transcribed/translated (IVTT) Bcl-2, Bcl-xL, or FLAG–Bcl-xS inserted (alkali resistant, integral) or loosely attached (alkali extractable, peripheral) to mitochondria or microsomes (pellet), or remaining in the supernatant after spinning off the organelles. (E) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of R6 cells transiently overexpressing Bcl-xL, FLAG-Bcl-xS, or Bcl-2 (green). Whereas both Bcl-xL and FLAG–Bcl-xS colocalize with the mitochondrial marker cytochrome c (Cyt.c, red), Bcl-2 colocalizes with the ER marker calnexin (red). Nuclei were stained with Hoechst 33342 (blue in the merge).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172731&req=5

fig1: Bcl-xL and Bcl-xS specifically localize to the MOM, whereas Bcl-2 resides on several membranes. (A) Anti–Bcl-2 and anti–Bcl-x Western blots of subcellular fractions from parental HEK 293 cells (endo) or HEK293 cells transiently overexpressing Bcl-2, Bcl-xL, or FLAG–Bcl-xS (exo). The mitochondrial fraction was obtained from the 1.4 M band and the heavy microsomal fraction from the 1.0 M band of the sucrose gradient (see Materials and methods). Light microsomes are the pellet of the 100,000 g spin. Purity of the fractions was checked with anti-grp78/Bip and anti-KDEL (microsomes) and anti–COX-VIc (mitochondria) antibodies. TAP(I-VI)–EGFP contains the first six membrane-spanning regions of the antigen peptide transporter I (TAP I) fused to EGFP. This protein specifically spans the ER membrane and is detected by anti-GFP Western blotting after transient transfection. (B) Anti–Bcl-x Western blots of mitochondrial matrix, inner membrane (mb), and outer membrane fractions of rat liver, HEK293 cells, and HEK293 cells transiently overexpressing Bcl-xL (HEK293/Bcl-xL). (C) Anti–Bcl-2 and anti–Bcl-x Western blots of mitochondria or microsomes from parental HEK293 cells or HEK293 cells overexpressing Bcl-2 or Bcl-xL or FLAG–Bcl-xS, extracted directly with detergent (total) or first treated with sodium carbonate (pH 12, peripheral) and then extracted with detergent (integral). (D) Autoradiography of [35S]methionine-labeled, in vitro–transcribed/translated (IVTT) Bcl-2, Bcl-xL, or FLAG–Bcl-xS inserted (alkali resistant, integral) or loosely attached (alkali extractable, peripheral) to mitochondria or microsomes (pellet), or remaining in the supernatant after spinning off the organelles. (E) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of R6 cells transiently overexpressing Bcl-xL, FLAG-Bcl-xS, or Bcl-2 (green). Whereas both Bcl-xL and FLAG–Bcl-xS colocalize with the mitochondrial marker cytochrome c (Cyt.c, red), Bcl-2 colocalizes with the ER marker calnexin (red). Nuclei were stained with Hoechst 33342 (blue in the merge).

Mentions: Endogenous and overexpressed Bcl-xL were enriched in mitochondria when purified subcellular fractions of HEK293 cells were analyzed on anti–Bcl-x immunoblots (Fig. 1 A). A protein band with a slightly higher molecular mass was detected in the light microsomal fraction of cells overexpressing Bcl-xL (Fig. 1 A). In certain cell types, Bcl-xL also appeared in the cytosol, especially when the protein was overexpressed (unpublished data). Immunoblots with organelle-specific marker antibodies revealed that the subcellular fractions were pure (Fig. 1 A). Nuclear fractions are not shown because they could not be reproducibly deprived of cosedimenting or aggregated mitochondria and unlysed cells. A further subfractionation of HEK293 and rat liver mitochondria showed that the major proportion of Bcl-xL resided in the MOM (Fig. 1 B). Endogenous Bcl-xL was stably integrated into the mitochondrial membrane because it could only be extracted with detergent (integral) (Fig. 1 C). By contrast, some of the overexpressed Bcl-xL was loosely (peripherally) attached to this membrane. When Bcl-xL was in vitro transcribed/translated and added to purified mitochondria, most of the protein was recovered in the mitochondrial pellet in a stably inserted form (Fig. 1 D). The pro-apoptotic splice variant Bcl-xS, which is identical to Bcl-xL but lacks the BH1/BH2 regions (Boise et al., 1993), also accumulated in mitochondria after ectopic expression in HEK293 cells (Fig. 1 A) and in vitro translation (Fig. 1 D) and inserted even better into the mitochondrial membrane than Bcl-xL (Fig. 1 C). The endogenous Bcl-xS protein could not be studied because it was not expressed at detectable levels in 10 different cell lines (unpublished data). Strikingly, in contrast to Bcl-xL and Bcl-xS, endogenous and overexpressed Bcl-2 were found in all intracellular membrane fractions (Fig. 1 A). The mitochondrial portion of Bcl-2 may be even overestimated, as some heavy microsomal membranes could not be entirely separated from mitochondria, as evidenced by the ER membrane–specific marker TAP(I-VI)–EGFP (Vos et al., 2000) (Fig. 1 A). This fraction did not contain the luminal grp78/Bip and KDEL proteins and may consist of microsomal membranes that are fused to mitochondria as recently proposed (Landolfi et al., 1998; Prinz et al., 2000). Bcl-2 integrated into the microsomal membrane in a similar way as Bcl-xL integrated into mitochondria. Although endogenous Bcl-2 was never cytosolic and stably inserted into membranes (Fig. 1, A and C), some of the overexpressed form loosely attached to microsomes (Fig. 1 C) and remained in the supernatant after in vitro transcription/translation (Fig. 1 D). Immunofluorescence analysis of R6 (Fig. 1 E) or HeLa cells (unpublished data) confirmed our in vitro data. Whereas Bcl-2 colocalized with the ER protein calnexin on nuclear/ER membrane structures, Bcl-xL and Bcl-xS were consistently immunodetected in filamentous structures that colocalized with the mitochondrial markers TOM20, COX, or cytochrome c (Fig. 1 E). Our data show that Bcl-xL/xS and Bcl-2 are primarily membrane-inserted proteins, but with different targeting specificities.


Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane.

Kaufmann T, Schlipf S, Sanz J, Neubert K, Stein R, Borner C - J. Cell Biol. (2003)

Bcl-xL and Bcl-xS specifically localize to the MOM, whereas Bcl-2 resides on several membranes. (A) Anti–Bcl-2 and anti–Bcl-x Western blots of subcellular fractions from parental HEK 293 cells (endo) or HEK293 cells transiently overexpressing Bcl-2, Bcl-xL, or FLAG–Bcl-xS (exo). The mitochondrial fraction was obtained from the 1.4 M band and the heavy microsomal fraction from the 1.0 M band of the sucrose gradient (see Materials and methods). Light microsomes are the pellet of the 100,000 g spin. Purity of the fractions was checked with anti-grp78/Bip and anti-KDEL (microsomes) and anti–COX-VIc (mitochondria) antibodies. TAP(I-VI)–EGFP contains the first six membrane-spanning regions of the antigen peptide transporter I (TAP I) fused to EGFP. This protein specifically spans the ER membrane and is detected by anti-GFP Western blotting after transient transfection. (B) Anti–Bcl-x Western blots of mitochondrial matrix, inner membrane (mb), and outer membrane fractions of rat liver, HEK293 cells, and HEK293 cells transiently overexpressing Bcl-xL (HEK293/Bcl-xL). (C) Anti–Bcl-2 and anti–Bcl-x Western blots of mitochondria or microsomes from parental HEK293 cells or HEK293 cells overexpressing Bcl-2 or Bcl-xL or FLAG–Bcl-xS, extracted directly with detergent (total) or first treated with sodium carbonate (pH 12, peripheral) and then extracted with detergent (integral). (D) Autoradiography of [35S]methionine-labeled, in vitro–transcribed/translated (IVTT) Bcl-2, Bcl-xL, or FLAG–Bcl-xS inserted (alkali resistant, integral) or loosely attached (alkali extractable, peripheral) to mitochondria or microsomes (pellet), or remaining in the supernatant after spinning off the organelles. (E) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of R6 cells transiently overexpressing Bcl-xL, FLAG-Bcl-xS, or Bcl-2 (green). Whereas both Bcl-xL and FLAG–Bcl-xS colocalize with the mitochondrial marker cytochrome c (Cyt.c, red), Bcl-2 colocalizes with the ER marker calnexin (red). Nuclei were stained with Hoechst 33342 (blue in the merge).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172731&req=5

fig1: Bcl-xL and Bcl-xS specifically localize to the MOM, whereas Bcl-2 resides on several membranes. (A) Anti–Bcl-2 and anti–Bcl-x Western blots of subcellular fractions from parental HEK 293 cells (endo) or HEK293 cells transiently overexpressing Bcl-2, Bcl-xL, or FLAG–Bcl-xS (exo). The mitochondrial fraction was obtained from the 1.4 M band and the heavy microsomal fraction from the 1.0 M band of the sucrose gradient (see Materials and methods). Light microsomes are the pellet of the 100,000 g spin. Purity of the fractions was checked with anti-grp78/Bip and anti-KDEL (microsomes) and anti–COX-VIc (mitochondria) antibodies. TAP(I-VI)–EGFP contains the first six membrane-spanning regions of the antigen peptide transporter I (TAP I) fused to EGFP. This protein specifically spans the ER membrane and is detected by anti-GFP Western blotting after transient transfection. (B) Anti–Bcl-x Western blots of mitochondrial matrix, inner membrane (mb), and outer membrane fractions of rat liver, HEK293 cells, and HEK293 cells transiently overexpressing Bcl-xL (HEK293/Bcl-xL). (C) Anti–Bcl-2 and anti–Bcl-x Western blots of mitochondria or microsomes from parental HEK293 cells or HEK293 cells overexpressing Bcl-2 or Bcl-xL or FLAG–Bcl-xS, extracted directly with detergent (total) or first treated with sodium carbonate (pH 12, peripheral) and then extracted with detergent (integral). (D) Autoradiography of [35S]methionine-labeled, in vitro–transcribed/translated (IVTT) Bcl-2, Bcl-xL, or FLAG–Bcl-xS inserted (alkali resistant, integral) or loosely attached (alkali extractable, peripheral) to mitochondria or microsomes (pellet), or remaining in the supernatant after spinning off the organelles. (E) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of R6 cells transiently overexpressing Bcl-xL, FLAG-Bcl-xS, or Bcl-2 (green). Whereas both Bcl-xL and FLAG–Bcl-xS colocalize with the mitochondrial marker cytochrome c (Cyt.c, red), Bcl-2 colocalizes with the ER marker calnexin (red). Nuclei were stained with Hoechst 33342 (blue in the merge).
Mentions: Endogenous and overexpressed Bcl-xL were enriched in mitochondria when purified subcellular fractions of HEK293 cells were analyzed on anti–Bcl-x immunoblots (Fig. 1 A). A protein band with a slightly higher molecular mass was detected in the light microsomal fraction of cells overexpressing Bcl-xL (Fig. 1 A). In certain cell types, Bcl-xL also appeared in the cytosol, especially when the protein was overexpressed (unpublished data). Immunoblots with organelle-specific marker antibodies revealed that the subcellular fractions were pure (Fig. 1 A). Nuclear fractions are not shown because they could not be reproducibly deprived of cosedimenting or aggregated mitochondria and unlysed cells. A further subfractionation of HEK293 and rat liver mitochondria showed that the major proportion of Bcl-xL resided in the MOM (Fig. 1 B). Endogenous Bcl-xL was stably integrated into the mitochondrial membrane because it could only be extracted with detergent (integral) (Fig. 1 C). By contrast, some of the overexpressed Bcl-xL was loosely (peripherally) attached to this membrane. When Bcl-xL was in vitro transcribed/translated and added to purified mitochondria, most of the protein was recovered in the mitochondrial pellet in a stably inserted form (Fig. 1 D). The pro-apoptotic splice variant Bcl-xS, which is identical to Bcl-xL but lacks the BH1/BH2 regions (Boise et al., 1993), also accumulated in mitochondria after ectopic expression in HEK293 cells (Fig. 1 A) and in vitro translation (Fig. 1 D) and inserted even better into the mitochondrial membrane than Bcl-xL (Fig. 1 C). The endogenous Bcl-xS protein could not be studied because it was not expressed at detectable levels in 10 different cell lines (unpublished data). Strikingly, in contrast to Bcl-xL and Bcl-xS, endogenous and overexpressed Bcl-2 were found in all intracellular membrane fractions (Fig. 1 A). The mitochondrial portion of Bcl-2 may be even overestimated, as some heavy microsomal membranes could not be entirely separated from mitochondria, as evidenced by the ER membrane–specific marker TAP(I-VI)–EGFP (Vos et al., 2000) (Fig. 1 A). This fraction did not contain the luminal grp78/Bip and KDEL proteins and may consist of microsomal membranes that are fused to mitochondria as recently proposed (Landolfi et al., 1998; Prinz et al., 2000). Bcl-2 integrated into the microsomal membrane in a similar way as Bcl-xL integrated into mitochondria. Although endogenous Bcl-2 was never cytosolic and stably inserted into membranes (Fig. 1, A and C), some of the overexpressed form loosely attached to microsomes (Fig. 1 C) and remained in the supernatant after in vitro transcription/translation (Fig. 1 D). Immunofluorescence analysis of R6 (Fig. 1 E) or HeLa cells (unpublished data) confirmed our in vitro data. Whereas Bcl-2 colocalized with the ER protein calnexin on nuclear/ER membrane structures, Bcl-xL and Bcl-xS were consistently immunodetected in filamentous structures that colocalized with the mitochondrial markers TOM20, COX, or cytochrome c (Fig. 1 E). Our data show that Bcl-xL/xS and Bcl-2 are primarily membrane-inserted proteins, but with different targeting specificities.

Bottom Line: Bcl-2 lacks the signal and therefore localizes to several intracellular membranes.The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM.These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, D-79106 Freiburg, Germany.

ABSTRACT
It is assumed that the survival factors Bcl-2 and Bcl-x(L) are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-x(L) is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-x(L) requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.

Show MeSH
Related in: MedlinePlus