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
MOM targeting also requires two basic residues downstream of the TM of Bcl-x and Bcl-2 accumulates on mitochondria by increasing the basicity surrounding its TM. (A and B) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of HeLa or R6 cells (as indicated) transiently transfected with FLAG-tagged Bcl-xL mutants that had their COOH-terminal basic residues exchanged to serines (A) or FLAG-tagged Bcl-2 mutants that contained additional basic residues at their COOH termini (B). In addition, the localization of COOH-terminal FLAG-tagged Bcl-xL is shown (Bcl-xL–FLAG). The mitochondrial colocalization markers are cytochrome c or TOM20 as indicated. (C) Anti-FLAG Western blots of subcellular fractions from HEK293 cells expressing the mutants described under A and B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172731&req=5

fig6: MOM targeting also requires two basic residues downstream of the TM of Bcl-x and Bcl-2 accumulates on mitochondria by increasing the basicity surrounding its TM. (A and B) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of HeLa or R6 cells (as indicated) transiently transfected with FLAG-tagged Bcl-xL mutants that had their COOH-terminal basic residues exchanged to serines (A) or FLAG-tagged Bcl-2 mutants that contained additional basic residues at their COOH termini (B). In addition, the localization of COOH-terminal FLAG-tagged Bcl-xL is shown (Bcl-xL–FLAG). The mitochondrial colocalization markers are cytochrome c or TOM20 as indicated. (C) Anti-FLAG Western blots of subcellular fractions from HEK293 cells expressing the mutants described under A and B.

Mentions: It was striking that the EGFP–AAGQEAFNA–TMB(Bcl-x) mutant still specifically associated with mitochondria whereas the EGFP–TMB(Bcl-x) mutant was fully cytosolic (Fig. 4 B). This suggested that the TMB of Bcl-x had a partial mitochondrial targeting capacity even though the preceding X domain did not contain the flanking basic amino acids. The TMB consists of the hydrophobic TM domain followed by two basic amino acids in the B domain (RK) (Fig. 2 A). To investigate whether these basic amino acids played a role in mitochondrial targeting, we mutated them in a FLAG-tagged version of Bcl-xL (to distinguish the mutants from endogenous Bcl-xL) (Fig. 2 B). FLAG-tagged Bcl-xL specifically localized to mitochondria like its nontagged counterpart (Fig. 6 A). By contrast, eliminating one positive charge of the B domain by mutating Arg232 or Lys233 to Ser was sufficient to prevent the specific mitochondrial association of Bcl-xL. Both the FLAG–Bcl-xL(SK) and FLAG–Bcl-xL(RS) mutants lost most of the mitochondria-specific localization pattern, stained the nuclear envelope and the ER membrane (Fig. 6 A), and resided on all intracellular membranes and in the cytosol in a similar way as Bcl-2 (Fig. 6 C). Moreover, the addition of an eight–amino acid FLAG peptide to the COOH terminus of Bcl-xL (Bcl-xL–FLAG; Fig. 2 B) led to a cytosolic distribution of Bcl-xL (Fig. 6 A), perhaps by neutralizing the extreme COOH-terminal RK amino acids. These data show that the mitochondrial sorting signal of Bcl-x does not only require two basic residues upstream (in the X domain) but also at least two basic residues downstream of its TM.


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)

MOM targeting also requires two basic residues downstream of the TM of Bcl-x and Bcl-2 accumulates on mitochondria by increasing the basicity surrounding its TM. (A and B) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of HeLa or R6 cells (as indicated) transiently transfected with FLAG-tagged Bcl-xL mutants that had their COOH-terminal basic residues exchanged to serines (A) or FLAG-tagged Bcl-2 mutants that contained additional basic residues at their COOH termini (B). In addition, the localization of COOH-terminal FLAG-tagged Bcl-xL is shown (Bcl-xL–FLAG). The mitochondrial colocalization markers are cytochrome c or TOM20 as indicated. (C) Anti-FLAG Western blots of subcellular fractions from HEK293 cells expressing the mutants described under A and B.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: MOM targeting also requires two basic residues downstream of the TM of Bcl-x and Bcl-2 accumulates on mitochondria by increasing the basicity surrounding its TM. (A and B) Anti–Bcl-2 and anti–Bcl-x immunofluorescence analysis of HeLa or R6 cells (as indicated) transiently transfected with FLAG-tagged Bcl-xL mutants that had their COOH-terminal basic residues exchanged to serines (A) or FLAG-tagged Bcl-2 mutants that contained additional basic residues at their COOH termini (B). In addition, the localization of COOH-terminal FLAG-tagged Bcl-xL is shown (Bcl-xL–FLAG). The mitochondrial colocalization markers are cytochrome c or TOM20 as indicated. (C) Anti-FLAG Western blots of subcellular fractions from HEK293 cells expressing the mutants described under A and B.
Mentions: It was striking that the EGFP–AAGQEAFNA–TMB(Bcl-x) mutant still specifically associated with mitochondria whereas the EGFP–TMB(Bcl-x) mutant was fully cytosolic (Fig. 4 B). This suggested that the TMB of Bcl-x had a partial mitochondrial targeting capacity even though the preceding X domain did not contain the flanking basic amino acids. The TMB consists of the hydrophobic TM domain followed by two basic amino acids in the B domain (RK) (Fig. 2 A). To investigate whether these basic amino acids played a role in mitochondrial targeting, we mutated them in a FLAG-tagged version of Bcl-xL (to distinguish the mutants from endogenous Bcl-xL) (Fig. 2 B). FLAG-tagged Bcl-xL specifically localized to mitochondria like its nontagged counterpart (Fig. 6 A). By contrast, eliminating one positive charge of the B domain by mutating Arg232 or Lys233 to Ser was sufficient to prevent the specific mitochondrial association of Bcl-xL. Both the FLAG–Bcl-xL(SK) and FLAG–Bcl-xL(RS) mutants lost most of the mitochondria-specific localization pattern, stained the nuclear envelope and the ER membrane (Fig. 6 A), and resided on all intracellular membranes and in the cytosol in a similar way as Bcl-2 (Fig. 6 C). Moreover, the addition of an eight–amino acid FLAG peptide to the COOH terminus of Bcl-xL (Bcl-xL–FLAG; Fig. 2 B) led to a cytosolic distribution of Bcl-xL (Fig. 6 A), perhaps by neutralizing the extreme COOH-terminal RK amino acids. These data show that the mitochondrial sorting signal of Bcl-x does not only require two basic residues upstream (in the X domain) but also at least two basic residues downstream of its TM.

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