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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 MOM- and ER-targeted proteins. (A) Amino acid sequences of MOM-targeted proteins whose COOH termini conform to the possible Bx0–9Bx0–2-TM-x0–1Bx0–6B consensus sequence or contain a high basicity at one end of their TM-like FLAG–Bcl-2(HKRK) (TOM20 and Bcl-w). (B) Amino acid sequences of proteins that contain only one basic residues at each end (Bcl-2 and Mcl-1), or basic residues at only one end of the TM and thus localize to extra-mitochondrial sites.
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fig7: Schematic representation of MOM- and ER-targeted proteins. (A) Amino acid sequences of MOM-targeted proteins whose COOH termini conform to the possible Bx0–9Bx0–2-TM-x0–1Bx0–6B consensus sequence or contain a high basicity at one end of their TM-like FLAG–Bcl-2(HKRK) (TOM20 and Bcl-w). (B) Amino acid sequences of proteins that contain only one basic residues at each end (Bcl-2 and Mcl-1), or basic residues at only one end of the TM and thus localize to extra-mitochondrial sites.

Mentions: Recent studies have shown that the targeting of tail- or tip-anchored proteins to the MOM depends on the length and hydrophobicity of a TM domain of 17–23 hydrophobic amino acids and a short hydrophilic sequence rich in basic residues either before or after the TM domain (McBride et al., 1992; Kuroda et al., 1998; Egan et al., 1999; Kanaji et al., 2000; Mihara, 2000; Horie et al., 2002). We however noted that these proteins contained basic amino acids at both ends of their TM (Fig. 7 A). Although a targeting role of positive charges on both sides have recently been suggested for the M11L protein of myxoma virus and, astonishingly, for Bcl-2, no mutagenesis analysis was performed to support this hypothesis (Everett et al., 2000). Here we combined both whole cell fluorescence and immunoblot analysis of pure organelle fractions to show that effective targeting of Bcl-x to the MOM requires at least two basic residues at both ends of the TM domain. Neither the hydrophobicity nor the length of the TM domain appear to play any role in targeting, as the TMs of Bcl-2 and Bcl-x have a similar degree of hydrophobicity and length despite different subcellular localizations. It may however be important at which distance the basic residues are located with respect to the TM domain, although this has not yet been directly tested. By comparing the MOM targeting sequences of several proteins, we propose that one basic residue is within one or two amino acids of the COOH and NH2 termini of the TM domain, respectively, whereas the second basic residue is up to six or nine amino acids distant from the first (Fig. 7 A). A comparison of the subcellular localization of the EGFP–RFNR–TMB(Bcl-x), EGFP–RAAR–TMB(Bcl-x), and EGFP–RR–TMB(Bcl-x) constructs (Fig. 4) further suggests that in the case of Bcl-x, the nature and number of the amino acids between the basic residues do not play a mitochondrial targeting role. On the basis of these data, a new putative consensus sequence for MOM signaling/anchoring is proposed in the form of Bx0–9Bx0–2-TM-x0–1Bx0–6B (where B stands for basic residues and x for any amino acid). This consensus is fulfilled by numerous proteins that are tail anchored in the MOM, such as the Bcl-2 family members Bcl-x, Bcl-B, BHRF-EBV, Nip3, Nix, and CED-9 as well as VAMP-1B, TOM-5, metaxin-1, myxoma viral M11L, monoamine oxidase A and B, and mitochondrial cytochrome b5 (Fig. 7 A). In the case of TOM20 and the Bcl-2 family member Bcl-w, which are mitochondrial proteins but display only one positive charge at one end of the TM, the other side contains a higher basicity (three to four basic residues) (Fig. 7 A), as seen with the mitochondria-targeted FLAG–Bcl-2(HKRK) construct (Fig. 6, B and C). By contrast, a variety of proteins that are targeted to the ER and other intracellular membranes lack the MOM targeting sequence, either because they contain basic residues at only one end of the TM (VAMP-1A, VAMP-2, VAMP-8, BET1, syntaxin 1A, microsomal cytochrome b5, and Bcl-2 homologue NR13) or do not have sufficient basicity at both ends (Bcl-2 and its homologue Mcl-1) (Fig. 7 B). Thus, the consensus sequence proposed here is more predictive for MOM sorting than the sequences previously reported. Exceptions are the pro-apoptotic Bcl-2 family members Bax and Bak, which contain basic residues only at one end of the TM but nevertheless localize to the MOM. These proteins probably require additional cellular factors to unleash the COOH-terminal mitochondrial targeting signal, which is folded back into the molecule after synthesis (Suzuki et al., 2000; unpublished data).


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 MOM- and ER-targeted proteins. (A) Amino acid sequences of MOM-targeted proteins whose COOH termini conform to the possible Bx0–9Bx0–2-TM-x0–1Bx0–6B consensus sequence or contain a high basicity at one end of their TM-like FLAG–Bcl-2(HKRK) (TOM20 and Bcl-w). (B) Amino acid sequences of proteins that contain only one basic residues at each end (Bcl-2 and Mcl-1), or basic residues at only one end of the TM and thus localize to extra-mitochondrial sites.
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Related In: Results  -  Collection

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

fig7: Schematic representation of MOM- and ER-targeted proteins. (A) Amino acid sequences of MOM-targeted proteins whose COOH termini conform to the possible Bx0–9Bx0–2-TM-x0–1Bx0–6B consensus sequence or contain a high basicity at one end of their TM-like FLAG–Bcl-2(HKRK) (TOM20 and Bcl-w). (B) Amino acid sequences of proteins that contain only one basic residues at each end (Bcl-2 and Mcl-1), or basic residues at only one end of the TM and thus localize to extra-mitochondrial sites.
Mentions: Recent studies have shown that the targeting of tail- or tip-anchored proteins to the MOM depends on the length and hydrophobicity of a TM domain of 17–23 hydrophobic amino acids and a short hydrophilic sequence rich in basic residues either before or after the TM domain (McBride et al., 1992; Kuroda et al., 1998; Egan et al., 1999; Kanaji et al., 2000; Mihara, 2000; Horie et al., 2002). We however noted that these proteins contained basic amino acids at both ends of their TM (Fig. 7 A). Although a targeting role of positive charges on both sides have recently been suggested for the M11L protein of myxoma virus and, astonishingly, for Bcl-2, no mutagenesis analysis was performed to support this hypothesis (Everett et al., 2000). Here we combined both whole cell fluorescence and immunoblot analysis of pure organelle fractions to show that effective targeting of Bcl-x to the MOM requires at least two basic residues at both ends of the TM domain. Neither the hydrophobicity nor the length of the TM domain appear to play any role in targeting, as the TMs of Bcl-2 and Bcl-x have a similar degree of hydrophobicity and length despite different subcellular localizations. It may however be important at which distance the basic residues are located with respect to the TM domain, although this has not yet been directly tested. By comparing the MOM targeting sequences of several proteins, we propose that one basic residue is within one or two amino acids of the COOH and NH2 termini of the TM domain, respectively, whereas the second basic residue is up to six or nine amino acids distant from the first (Fig. 7 A). A comparison of the subcellular localization of the EGFP–RFNR–TMB(Bcl-x), EGFP–RAAR–TMB(Bcl-x), and EGFP–RR–TMB(Bcl-x) constructs (Fig. 4) further suggests that in the case of Bcl-x, the nature and number of the amino acids between the basic residues do not play a mitochondrial targeting role. On the basis of these data, a new putative consensus sequence for MOM signaling/anchoring is proposed in the form of Bx0–9Bx0–2-TM-x0–1Bx0–6B (where B stands for basic residues and x for any amino acid). This consensus is fulfilled by numerous proteins that are tail anchored in the MOM, such as the Bcl-2 family members Bcl-x, Bcl-B, BHRF-EBV, Nip3, Nix, and CED-9 as well as VAMP-1B, TOM-5, metaxin-1, myxoma viral M11L, monoamine oxidase A and B, and mitochondrial cytochrome b5 (Fig. 7 A). In the case of TOM20 and the Bcl-2 family member Bcl-w, which are mitochondrial proteins but display only one positive charge at one end of the TM, the other side contains a higher basicity (three to four basic residues) (Fig. 7 A), as seen with the mitochondria-targeted FLAG–Bcl-2(HKRK) construct (Fig. 6, B and C). By contrast, a variety of proteins that are targeted to the ER and other intracellular membranes lack the MOM targeting sequence, either because they contain basic residues at only one end of the TM (VAMP-1A, VAMP-2, VAMP-8, BET1, syntaxin 1A, microsomal cytochrome b5, and Bcl-2 homologue NR13) or do not have sufficient basicity at both ends (Bcl-2 and its homologue Mcl-1) (Fig. 7 B). Thus, the consensus sequence proposed here is more predictive for MOM sorting than the sequences previously reported. Exceptions are the pro-apoptotic Bcl-2 family members Bax and Bak, which contain basic residues only at one end of the TM but nevertheless localize to the MOM. These proteins probably require additional cellular factors to unleash the COOH-terminal mitochondrial targeting signal, which is folded back into the molecule after synthesis (Suzuki et al., 2000; unpublished data).

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