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
Two basic amino acids of the X domain and the TMB of Bcl-x compose a signal that targets EGFP to mitochondria. (A) Whole cell GFP fluorescence analysis of R6 cells transiently transfected with EGFP fused to various COOH-terminal mutants of Bcl-x and Bcl-2. The amino acid sequences of the mutants are described in Fig. 2 C. The mitochondrial colocalization marker is cytochrome c. Point mutations are underlined. (B) Anti-GFP Western blots of subcellular fractions from HEK293 cells expressing the various EGFP mutants. (C) Sodium carbonate extraction, as described in legend to Fig. 1 C, of mitochondria or microsomes isolated from HEK cells transfected with three selected EGFP mutants.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172731&req=5

fig4: Two basic amino acids of the X domain and the TMB of Bcl-x compose a signal that targets EGFP to mitochondria. (A) Whole cell GFP fluorescence analysis of R6 cells transiently transfected with EGFP fused to various COOH-terminal mutants of Bcl-x and Bcl-2. The amino acid sequences of the mutants are described in Fig. 2 C. The mitochondrial colocalization marker is cytochrome c. Point mutations are underlined. (B) Anti-GFP Western blots of subcellular fractions from HEK293 cells expressing the various EGFP mutants. (C) Sodium carbonate extraction, as described in legend to Fig. 1 C, of mitochondria or microsomes isolated from HEK cells transfected with three selected EGFP mutants.

Mentions: To investigate whether the TMBs of Bcl-2 and Bcl-xL/xS alone were sufficient for membrane targeting, they were separately fused to the COOH-terminal end of EGFP (Fig. 2 C). To our surprise, both the EGFP–TMB(Bcl-2) and EGFP–TMB(Bcl-x) proteins displayed a diffuse staining by whole cell fluorescence and mainly localized to a cytosolic fraction (Fig. 4, A and B), suggesting a role for additional amino acid sequences in Bcl-x and Bcl-2 for membrane targeting. The region preceding the TMBs, called the X domain, has no sequence homology between Bcl-2 and Bcl-x (Fig. 2 A) and thus could be responsible for the distinct subcellular targeting of the two proteins. Introduction of the X domain of Bcl-x into the EGFP–TMB(Bcl-x) construct (EGFP–X–TMB[Bcl-x]) or half of the X domain of Bcl-2 (amino acids WLSLK) into the EGFP–TMB(Bcl-2) construct (EGFP–X/2–TMB[Bcl-2]) (Fig. 2, A and C) gave identical targeting specificities as for wild-type Bcl-xL/xS or Bcl-2, respectively (compare Fig. 4, A and B, to Fig. 1, A and E). The EGFP–X/2–TMB(Bcl-2) protein was immunodetected in all membrane fractions and produced a strong nuclear envelope/ER staining (Fig. 4, A and B). Adding the entire X domain and the preceding BH2 domain of Bcl-2 (EGFP–BH2X–TMB[Bcl-2]) did not alter its ubiquitous membrane distribution. By contrast, the EGFP–X–TMB(Bcl-x) protein was only found in a mitochondrial fraction (Fig. 4 B) and was specifically targeted to elongated, mitochondrial structures (Fig. 4 A). Both the EGFP–BH2X–TMB(Bcl-2) and the EGFP–X–TMB(Bcl-x) proteins were difficult to extract from membranes by alkali treatment, indicating that the X domains directed the stable membrane insertion of the TMBs (Fig. 4 C). Consistent with a crucial role of the X domain of Bcl-x in mitochondrial targeting, Bcl-xL lacking this domain (Bcl-xLΔX) (Fig. 2 B) was found to reside majorly in the cytosol, despite the fact that it retained its TMB (Fig. 5 C). These data show that in both Bcl-2 and Bcl-x, the X domain cooperates with the TMB for membrane association and stable insertion. In Bcl-x, it additionally constitutes a mitochondrial sorting signal.


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)

Two basic amino acids of the X domain and the TMB of Bcl-x compose a signal that targets EGFP to mitochondria. (A) Whole cell GFP fluorescence analysis of R6 cells transiently transfected with EGFP fused to various COOH-terminal mutants of Bcl-x and Bcl-2. The amino acid sequences of the mutants are described in Fig. 2 C. The mitochondrial colocalization marker is cytochrome c. Point mutations are underlined. (B) Anti-GFP Western blots of subcellular fractions from HEK293 cells expressing the various EGFP mutants. (C) Sodium carbonate extraction, as described in legend to Fig. 1 C, of mitochondria or microsomes isolated from HEK cells transfected with three selected EGFP mutants.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Two basic amino acids of the X domain and the TMB of Bcl-x compose a signal that targets EGFP to mitochondria. (A) Whole cell GFP fluorescence analysis of R6 cells transiently transfected with EGFP fused to various COOH-terminal mutants of Bcl-x and Bcl-2. The amino acid sequences of the mutants are described in Fig. 2 C. The mitochondrial colocalization marker is cytochrome c. Point mutations are underlined. (B) Anti-GFP Western blots of subcellular fractions from HEK293 cells expressing the various EGFP mutants. (C) Sodium carbonate extraction, as described in legend to Fig. 1 C, of mitochondria or microsomes isolated from HEK cells transfected with three selected EGFP mutants.
Mentions: To investigate whether the TMBs of Bcl-2 and Bcl-xL/xS alone were sufficient for membrane targeting, they were separately fused to the COOH-terminal end of EGFP (Fig. 2 C). To our surprise, both the EGFP–TMB(Bcl-2) and EGFP–TMB(Bcl-x) proteins displayed a diffuse staining by whole cell fluorescence and mainly localized to a cytosolic fraction (Fig. 4, A and B), suggesting a role for additional amino acid sequences in Bcl-x and Bcl-2 for membrane targeting. The region preceding the TMBs, called the X domain, has no sequence homology between Bcl-2 and Bcl-x (Fig. 2 A) and thus could be responsible for the distinct subcellular targeting of the two proteins. Introduction of the X domain of Bcl-x into the EGFP–TMB(Bcl-x) construct (EGFP–X–TMB[Bcl-x]) or half of the X domain of Bcl-2 (amino acids WLSLK) into the EGFP–TMB(Bcl-2) construct (EGFP–X/2–TMB[Bcl-2]) (Fig. 2, A and C) gave identical targeting specificities as for wild-type Bcl-xL/xS or Bcl-2, respectively (compare Fig. 4, A and B, to Fig. 1, A and E). The EGFP–X/2–TMB(Bcl-2) protein was immunodetected in all membrane fractions and produced a strong nuclear envelope/ER staining (Fig. 4, A and B). Adding the entire X domain and the preceding BH2 domain of Bcl-2 (EGFP–BH2X–TMB[Bcl-2]) did not alter its ubiquitous membrane distribution. By contrast, the EGFP–X–TMB(Bcl-x) protein was only found in a mitochondrial fraction (Fig. 4 B) and was specifically targeted to elongated, mitochondrial structures (Fig. 4 A). Both the EGFP–BH2X–TMB(Bcl-2) and the EGFP–X–TMB(Bcl-x) proteins were difficult to extract from membranes by alkali treatment, indicating that the X domains directed the stable membrane insertion of the TMBs (Fig. 4 C). Consistent with a crucial role of the X domain of Bcl-x in mitochondrial targeting, Bcl-xL lacking this domain (Bcl-xLΔX) (Fig. 2 B) was found to reside majorly in the cytosol, despite the fact that it retained its TMB (Fig. 5 C). These data show that in both Bcl-2 and Bcl-x, the X domain cooperates with the TMB for membrane association and stable insertion. In Bcl-x, it additionally constitutes a mitochondrial sorting signal.

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