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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.

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Schematic representation of Bcl-2, Bcl-xL, Bcl-xS, their mutants, and the EGFP fusion constructs. Schematic structure and amino acid sequences of (A) the COOH-terminal parts of wild-type Bcl-2 (yellow) and Bcl-xL/xS (blue), including the 19–amino acid-long TM domain, flanked by one to two basic amino acids at one end (B) and the X or X/2 domain (half of the X domain) at the other end (basic amino acids are numbered and indicated in bold); (B) the COOH-terminal parts of Bcl-2 and Bcl-xL/xS mutants (point mutations and insertions are underlined); (C) the COOH-terminal mutants of Bcl-2 and Bcl-x fused to the COOH terminus of EGFP.
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fig2: Schematic representation of Bcl-2, Bcl-xL, Bcl-xS, their mutants, and the EGFP fusion constructs. Schematic structure and amino acid sequences of (A) the COOH-terminal parts of wild-type Bcl-2 (yellow) and Bcl-xL/xS (blue), including the 19–amino acid-long TM domain, flanked by one to two basic amino acids at one end (B) and the X or X/2 domain (half of the X domain) at the other end (basic amino acids are numbered and indicated in bold); (B) the COOH-terminal parts of Bcl-2 and Bcl-xL/xS mutants (point mutations and insertions are underlined); (C) the COOH-terminal mutants of Bcl-2 and Bcl-x fused to the COOH terminus of EGFP.

Mentions: What determines the specific targeting of Bcl-xL/xS to the MOM and why is Bcl-2 incapable of doing so? Targeting sequences for the MOM have recently been identified in proteins of the TOM complex, monoamine oxidase A/B, VAMP-1B, and members of the Bcl-2 family (for reviews see Mihara, 2000; Wattenberg and Lithgow, 2001). These sequences are present at either the NH2 or COOH termini of the proteins and consist of a hydrophobic, α-helical TM region followed by one or two basic amino acids (TMB). Both Bcl-xL/xS and Bcl-2 contain a typical TMB domain at their COOH terminus (Fig. 2 A). To investigate the role of the TMB of Bcl-xL/xS and Bcl-2 in (mitochondrial) membrane targeting, this region was deleted (Fig. 2 B) and the tailless proteins transiently expressed in R6, HeLa, and HEK293 cells. Bcl-2ΔTMB, Bcl-xLΔTMB, and FLAG-tagged Bcl-xSΔTMB proteins were all immunodetected in cytosolic fractions and exhibited a diffuse cellular staining (Fig. 3, A and C). For Bcl-2ΔTMB, we also noticed a staining of the nuclear envelope (Fig. 3 C), and part of the protein copurified with a light microsomal fraction (Fig. 3 A). In addition, a portion of in vitro–translated Bcl-2ΔTMB was recovered in the pellet after incubation with microsomes (Fig. 3 B). However, the protein was only peripherally attached to membranes (Fig. 3 A), indicating that its membrane association was possibly a side effect of overexpression. Bcl-xLΔTMB and FLAG–Bcl-xSΔTMB were even less detected in membrane fractions than Bcl-2ΔTMB (Fig. 3, A and B), and the type of membrane was microsomal rather than mitochondrial, indicating that the tailless proteins lacked specific MOM targeting (Fig. 3 A). These data suggest that the TMB region is crucial for effective membrane insertion of all three proteins.


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)

Schematic representation of Bcl-2, Bcl-xL, Bcl-xS, their mutants, and the EGFP fusion constructs. Schematic structure and amino acid sequences of (A) the COOH-terminal parts of wild-type Bcl-2 (yellow) and Bcl-xL/xS (blue), including the 19–amino acid-long TM domain, flanked by one to two basic amino acids at one end (B) and the X or X/2 domain (half of the X domain) at the other end (basic amino acids are numbered and indicated in bold); (B) the COOH-terminal parts of Bcl-2 and Bcl-xL/xS mutants (point mutations and insertions are underlined); (C) the COOH-terminal mutants of Bcl-2 and Bcl-x fused to the COOH terminus of EGFP.
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Related In: Results  -  Collection

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fig2: Schematic representation of Bcl-2, Bcl-xL, Bcl-xS, their mutants, and the EGFP fusion constructs. Schematic structure and amino acid sequences of (A) the COOH-terminal parts of wild-type Bcl-2 (yellow) and Bcl-xL/xS (blue), including the 19–amino acid-long TM domain, flanked by one to two basic amino acids at one end (B) and the X or X/2 domain (half of the X domain) at the other end (basic amino acids are numbered and indicated in bold); (B) the COOH-terminal parts of Bcl-2 and Bcl-xL/xS mutants (point mutations and insertions are underlined); (C) the COOH-terminal mutants of Bcl-2 and Bcl-x fused to the COOH terminus of EGFP.
Mentions: What determines the specific targeting of Bcl-xL/xS to the MOM and why is Bcl-2 incapable of doing so? Targeting sequences for the MOM have recently been identified in proteins of the TOM complex, monoamine oxidase A/B, VAMP-1B, and members of the Bcl-2 family (for reviews see Mihara, 2000; Wattenberg and Lithgow, 2001). These sequences are present at either the NH2 or COOH termini of the proteins and consist of a hydrophobic, α-helical TM region followed by one or two basic amino acids (TMB). Both Bcl-xL/xS and Bcl-2 contain a typical TMB domain at their COOH terminus (Fig. 2 A). To investigate the role of the TMB of Bcl-xL/xS and Bcl-2 in (mitochondrial) membrane targeting, this region was deleted (Fig. 2 B) and the tailless proteins transiently expressed in R6, HeLa, and HEK293 cells. Bcl-2ΔTMB, Bcl-xLΔTMB, and FLAG-tagged Bcl-xSΔTMB proteins were all immunodetected in cytosolic fractions and exhibited a diffuse cellular staining (Fig. 3, A and C). For Bcl-2ΔTMB, we also noticed a staining of the nuclear envelope (Fig. 3 C), and part of the protein copurified with a light microsomal fraction (Fig. 3 A). In addition, a portion of in vitro–translated Bcl-2ΔTMB was recovered in the pellet after incubation with microsomes (Fig. 3 B). However, the protein was only peripherally attached to membranes (Fig. 3 A), indicating that its membrane association was possibly a side effect of overexpression. Bcl-xLΔTMB and FLAG–Bcl-xSΔTMB were even less detected in membrane fractions than Bcl-2ΔTMB (Fig. 3, A and B), and the type of membrane was microsomal rather than mitochondrial, indicating that the tailless proteins lacked specific MOM targeting (Fig. 3 A). These data suggest that the TMB region is crucial for effective membrane insertion of all three proteins.

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