Article |
Address correspondence to C. Borner, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Zentrale Klinische Forschung, Breisacherstrasse 66, D-79106 Freiburg, Germany. Tel.: 49-761-270-6217. Fax: 49-761-270-6218. E-mail: borner{at}ukl.uni-freiburg.de
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Abstract |
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Key Words: apoptosis; Bcl-2; Bcl-xL; mitochondria; ER
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Introduction |
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Whereas Bax-like factors increase mitochondrial permeability, Bcl-2like factors block this process (Antonsson, 2001; Cory and Adams, 2002). The two protein classes can neutralize each other by heterodimerization but this does not entirely explain their function (Knudson and Korsmeyer, 1997). Recent studies showed that the BH3-only proteins are required for the apoptotic regulation of Bcl-2 and Bax-like factors (Puthalakath and Strasser, 2002). These proteins act as "sensors" for apoptotic stimuli at different locations within the cells. In response to a defined stimulus, a particular BH3-only protein is modified by proteolysis or posttranslational modification, leaves its intracellular site, and travels to the mitochondrial membrane where it can choose two fates. Either it interacts, via its BH3 domain, with Bcl-2like survival factors to release a pro-apoptotic protein, or it interacts with Bax-like factors to stimulate their oligomerization and/or membrane insertion. In both cases, Bax and Bak are activated to trigger MOM perforation by a mechanism that has yet to be identified. On the other hand, Bcl-2 and Bcl-xL inhibit cell death when sufficiently expressed to sequester BH3-only proteins, Bax-like factors, and other pro-apoptotic molecules (Cory and Adams, 2002).
It is thought that Bcl-2 and Bcl-xL are redundant in their capacity to protect cells from apoptosis (Chao et al., 1995). Both proteins block BH3-only and Bax/Bak-mediated MOM perforation and may therefore primarily function on mitochondria. However, Bcl-2 also localizes to the nuclear envelope and the membrane of the ER (Krajewski et al., 1993; Givol et al., 1994; Janiak et al., 1994; Lithgow et al., 1994; Conus et al., 2000b), where it can theoretically sequester pro-apoptotic molecules in a similar way as on mitochondria. Indeed, if Bcl-2 is artificially targeted to the ER, it still protects cells from apoptosis (Zhu et al., 1996; Hacki et al., 2000). A recent study revealed that specific targeting of Bcl-2 to mitochondria converts it into a pro-apoptotic molecule (Wang et al., 2001), suggesting that mitochondria may not be the preferred site for the survival action of Bcl-2.
Both Bcl-2 and Bcl-xL are tail-anchored proteins, i.e., they contain a COOH-terminal hydrophobic helix that functions as a membrane insertion device (transmembrane [TM] domain) (Chen-Levy and Cleary, 1990; Nguyen et al., 1993; Janiak et al., 1994; Kim et al., 1997). Proteins that are tail anchored to the MOM or the ER, such as the MOM translocases TOM22 (Egan et al., 1999) and TOM5 (Horie et al., 2002) or the microsomal form of cytochrome b5 (De Silvestris et al., 1995), use their TM anchor also as a targeting signal. Although targeting signals for the translocation of proteins across the ER membrane or the inner mitochondrial membrane are well known (Neupert, 1997), those that target proteins to the MOM have only recently begun to be unveiled. A putative targeting signal for the MOM may be a hydrophobic TM region with a particular length and hydrophobicity followed by one to two basic amino acids (TMB) (Mihara, 2000; Wattenberg and Lithgow, 2001). As both Bcl-2 and Bcl-xL contain one to two basic amino acids after their TM region, it was proposed that they are both targeted to the MOM (Kuroda et al., 1998; Everett et al., 2000). However, localization studies have been mostly performed under nonphysiological conditions where Bcl-2 and Bcl-xL were overexpressed. It has therefore remained elusive whether endogenous Bcl-2 and Bcl-xL distribute evenly or are specifically targeted to the MOM. Moreover, there has not been a systematic analysis on the signal sequence that directs these proteins to organelles.
Here we perform an extensive mutagenesis analysis in the COOH terminus of Bcl-2 and Bcl-xL. We show that Bcl-xL contains a particular mitochondrial signal sequence in this region whereas Bcl-2 does not. This signal requires two basic amino acids at both ends of the TM domain. Importantly, we show that not only overexpressed but also endogenous Bcl-xL and Bcl-2 localize to different subcellular compartments. It is proposed that Bcl-xL specifically acts on mitochondria whereas Bcl-2 can also control ER events associated with apoptosis.
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Results |
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The TMB of Bcl-2 can only partially collaborate with the X domain of Bcl-x for mitochondrial sorting
Considering the possibility that the TMB of Bcl-2 may have mitochondrial sorting capacity when combined with the correct X domain, we generated a EGFP fusion protein that contained the X domain of Bcl-x and the TMB of Bcl-2. This EGFPX(Bcl-x)TMB(Bcl-2) fusion protein was better targeted to the mitochondrial fraction (Fig. 4 B) and localized more specifically to mitochondrial structures (Fig. 4 A) than the EGFPX/2TMB(Bcl-2) or EGFPBH2XTMB(Bcl-2) constructs (which contained the X domain of Bcl-2). However, the protein still appeared in heavy microsomes as well as in the cytosol (Fig. 4 B). Similar results were obtained when the X(Bcl-x)TMB(Bcl-2) region was fused to Bcl-xL (Bcl-xL[X(Bcl-x)TMB(Bcl-2)]) or to Bcl-2 (Bcl-2[X(Bcl-x)TMB(Bcl-2)]). The chimeric proteins colocalized with mitochondrial markers (yellow in the merge) and were partially enriched in mitochondrial fractions, but significant amounts of the proteins resided in other cellular compartments (punctated green in the merge), such as on microsomes and in the cytoplasm (Fig. 5, B and C). These data suggest that although the X domain of Bcl-x can collaborate with the TMB of Bcl-2 for mitochondrial targeting, effective sorting requires the TMB of Bcl-x.
Two basic residues preceding the TMB of Bcl-x compose with the TMB a mitochondrial targeting signal, but additional basic residues in the X domain stabilize the signal
Which amino acids in the X domain of Bcl-x were crucial to cooperate with the TMB for mitochondrial targeting? Because this domain contains four basic residues (as compared with only one in Bcl-2) (Fig. 2 A), the role of these positive charges was investigated. First, the basic amino acid that lies closest to the TMB domain (Arg212) was introduced into the EGFPTMB(Bcl-x) construct (Fig. 2 C). Compared with the EGFPTMB(Bcl-x) protein, which was entirely cytosolic, EGFPRTMB(Bcl-x) partially localized to mitochondrial structures (Fig. 4 A) and was retained in a mitochondrial fraction (Fig. 4 B). However, most of the fusion protein still resided in the cytosol and on light microsomes (Fig. 4 B), and its membrane association was rather peripheral (Fig. 4 C). This is in agreement with the prominent cytoplasmic localization of the Bcl-xLX mutant, which lacked the X domain but retained Arg212 and the TMB (Fig. 2 B; Fig. 5 C). Thus, Arg212 was insufficient for specific mitochondrial targeting and membrane insertion. The preceding region was then extended to the next basic residue (Arg209) (Fig. 2, A and C). The respective EGFPRFNRTMB(Bcl-x) fusion protein displayed a striking mitochondrial localization pattern (Fig. 4 A) and predominantly resided in mitochondrial membranes in a stably inserted form (Fig. 4 B; unpublished data). Despite that, a low amount of the protein remained in cytosolic and microsomal fractions when compared with EGFPXTMB(Bcl-x) (Fig. 4 B) or Bcl-xL (Fig. 1 A). Additional amino acids in the X region, in particular the first two basic residues (Arg204 and Lys205), were studied for their capacity to assist mitochondrial targeting (Fig. 2 A). As compared with EGFPXTMB(Bcl-x), which was entirely mitochondrial, the EGFPAAGQERFNRTMB(Bcl-x), where Arg204 and Lys205 were mutated to Ala, similarly appeared in microsomes and the cytosol as EGFPRFNRTMB(Bcl-x) (Fig. 4 B). These results showed that Arg204 and Lys205 of the X domain slightly increased the mitochondrial sorting property of RFNRTMB(Bcl-x). Consistent with this notion, an EGFPXTMB(Bcl-x) fusion protein that had all basic residues exchanged to alanine (EGFPAAGQEAFNATMB[Bcl-x]) lost most of its mitochondrial targeting function and became largely cytosolic (Fig. 4 B). In summary, although the RFNR sequence is both required and sufficient to cooperate with the TMB for mitochondrial targeting, Arg204 and Lys205 of the X domain slightly enhance this targeting.
To prove that the two Arg, and not the Phe/Asn sequence, in RFNR conferred the mitochondrial signaling, we mutated these amino acids (Fig. 2 C). EGFP fusion proteins with both Phe and Asn deleted (EGFPRRTMB[Bcl-x]) or exchanged to Ala (EGFPRAARTMB[Bcl-x]) localized to mitochondria with a similar efficiency as the RFNR construct (Fig. 4, A and B). By contrast, EGFP fusions that had one or both Arg exchanged to Ala (EGFPAFNRTMB[Bcl-x], EGFPRFNATMB[Bcl-x], and EGFPAFNATMB[Bcl-x]) lost most of their mitochondrial association and became cytosolic and microsomal (Fig. 4, A and B; unpublished data). These mutants also showed a cytoplasmic clustering (Fig. 4 A) that was lethal to cells 48 h after transfection (unpublished data). Thus, the two basic residues, and not the amino acids between these residues, cooperate with the TMB of Bcl-x for mitochondrial sorting.
The mitochondrial sorting signal of Bcl-x also requires at least two basic residues downstream of the TM
It was striking that the EGFPAAGQEAFNATMB(Bcl-x) mutant still specifically associated with mitochondria whereas the EGFPTMB(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 FLAGBcl-xL(SK) and FLAGBcl-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 eightamino acid FLAG peptide to the COOH terminus of Bcl-xL (Bcl-xLFLAG; 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.
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Discussion |
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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 1723 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 EGFPRFNRTMB(Bcl-x), EGFPRAARTMB(Bcl-x), and EGFPRRTMB(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 Bx09Bx02-TM-x01Bx06B (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 FLAGBcl-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).
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Kanaji et al. (2000) reported that the TM region of TOM20 functions by itself as a signal-anchor sequence to target GFP fusions to the ER. Bcl-2 and Bcl-xL both have a TM with similar hydophobicity as TOM20. Surprisingly, however, these hydrophobic TM domains were insufficient for membrane targeting and insertion. Even the addition of a basic residue in front of the TMB (EGFPKTMB[Bcl-2], data not shown; or EGFPRTMB[Bcl-x], Fig. 4, A and B) did not majorly enhance membrane targeting, and the small amount of protein that was membrane associated was loosely attached. Only when the sequence included the targeting motif, i.e., either the two basic residues NH2-terminal to the TMB of Bcl-xL or the X domain ahead of the TMB of Bcl-2, were the respective proteins stably inserted into membranes. This supports the notion that targeting and anchoring are intimately linked, i.e., the same sequence functions as a targeting and insertion device (for review see Mihara, 2000).
Quantitative immunoelectron microscopy revealed that, depending on the cell type, 5585% of Bcl-2 molecules are localized to the ER, and the remainder are inserted into the MOM of mammalian cells (Krajewski et al., 1993; Lithgow et al., 1994). In yeast, a fusion protein between the last 33 amino acids of Bcl-2 and a reporter protein is even exclusively targeted to the ER (Egan et al., 1999). We were unable to find any sequences in Bcl-2 that would target this protein specifically to any membrane in rat and human cells, thus excluding the possibility that this protein contains multiple targeting signals, as had been suggested (Lan et al., 2000). Moreover, in contrast to other ER-bound proteins such as VAMP-1A, the binding of Bcl-2 and microsomal cytochrome b5 to ER microsomes has been reported to be spontaneous and nonsaturable (Kim et al., 1997). This strongly suggests that Bcl-2 may not be targeted to this membrane but distributes on all intracellular membranes depending on the kinetic preference for one membrane over another (Wattenberg and Lithgow, 2001). Nuclear outer membrane and ER staining of Bcl-2 by immunofluorescence (Fig. 1 E; Fig. 4 A) may therefore simply reflect the fact that this endomembrane system has the largest surface in the cell. It was noticed that the two basic amino acids located after the TM domain of Bcl-2, His238-Lys239, may not be sufficient for MOM targeting, and that some portion of the protein may leak out of the mitochondrial transport apparatus, allowing Bcl-2 to be transported to or associated with the ER or other membranes (Kuroda et al., 1998). We show here that this potential "leaking out" can be prevented by increasing the basicity at the COOH terminus. Thus, the COOH terminus of Bcl-2 can be converted into a signal for the MOM, but usually acts as a nontargeting device in the wild-type protein.
An obvious question is whether Bcl-2 and Bcl-xL exert different functions depending on their intracellular site of localization. When Bcl-2 was specifically targeted to mitochondria (e.g., Bcl-2[X-TMB(Bcl-x)]) and tested for its ability to block staurosporine- or brefeldin A/cycloheximide-induced apoptosis of HeLa cells, it was found that the anti-apoptotic activity of the mutant was only slightly reduced when compared with the wild-type forms (unpublished data). Similarly, Bcl-xL targeted to the ER via the tail of the microsomal cytochrome b5 (unpublished data), or Bcl-xL carrying the COOH terminus of Bcl-2 (Bcl-xL[X-TMB (Bcl-2)]), exhibited a survival activity that was nearly as efficient as that of the wild-type Bcl-xL. Also, consistent with previous results (Borner et al., 1994), cytoplasmic mutants of Bcl-2 and Bcl-xL still possess partial anti-apoptotic activity although this continued activity may be due to a minor association between the tailless mutants and membranes, as shown in Fig. 3. Thus, our data support the notion that Bcl-2 and Bcl-xL are exchangeable survival factors that, independent of their subcellular localization, repress a common cell death pathway (Chao et al., 1995). Whereas previous reports have proposed nonredundant functions of Bcl-2 and Bcl-xL (Gottschalk et al., 1994; Coulson et al., 1999; Yuste et al., 2002), these studies were all undertaken using cells that overexpressed these proteins to high levels. Here we have begun to study the subcellular localization of the endogenous proteins. It will now be crucial to examine whether endogenous Bcl-xL exerts a mitochondria-specific function that cannot be replaced by Bcl-2, or whether endogenous Bcl-2 regulates ER-associated events of apoptosis that are not affected by Bcl-xL. Such organelle-specific actions may include posttranslational modifications such as the recently published deamidation of Bcl-xL, which inactivates the endogenous protein in response to DNA damaging agents and does not occur on Bcl-2 (Deverman et al., 2002). Studies using Bcl-x and Bcl-2 knockout mice have further shown that these proteins play nonredundant roles in tissues and cells (Veis et al., 1993; Motoyama et al., 1995). Although this may simply reflect a difference in tissue distribution, it could also be a consequence of distinct subcellular localizations and/or molecular mechanisms to oppose cell death of Bcl-2 and Bcl-xL. To solve this issue, one would for example need to express Bcl-2 on mitochondria at similar levels as Bcl-xL in a Bcl-xnull tissue and examine whether this now rescues the knockout phenotype. Irrespective of the result, the data presented here improve our understanding of how proteins are specifically targeted to the MOM and how they differ from proteins that accumulate on other intracellular membranes.
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Materials and methods |
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Protein expression and subcellular fractionation
Human embryonic kidney cells (HEK293) were grown on 100 mm until 80% confluent and then transfected with 10 µg of plasmid DNA using 25 µl of Superfect (QIAGEN) as described by the manufacturer. After 36 h, the SuperfectDNA complexes were removed and the cells were cultured for another 42 h. 35 x 107 HEK293 cells were homogenized in MSH buffer (210 mM mannitol, 70 mM sucrose, 20 mM Hepes, 1 mM EDTA, pH 7.4) plus protease inhibitors, and nuclei and cellular debris were removed by centrifugation at 500 g for 5 min. The post-nuclear supernatant was centrifuged at 5,100 g for 10 min to obtain the crude mitochondrial pellet. For further purification, the crude mitochondria were laid on top of a 12 M linear sucrose gradient and centrifuged at 52,000 g for 90 min in a SW41 rotor (Beckman Coulter). Purified mitochondria, which banded at a region corresponding to 1.4 M sucrose, were carefully collected and diluted to 0.25 M sucrose before centrifugation at 30,000 g for 30 min. The resulting pellets were solubilized in buffer H (20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 6 mM ß-mercaptoethanol, pH 7.5) containing 1% SDS. The band at 1.0 M sucrose, which corresponded to heavy microsomes, was similarly processed. Centrifugation of the post-mitochondrial supernatant at 100,000 g for 60 min yielded the light microsomal pellet and the cytosolic supernatant. Cytosols were immediately frozen; light microsomes were resuspended in buffer H containing 1% SDS. All fractions were tested for their purity by immunoblot analysis using antibodies against organelle-specific proteins.
Submitochondrial fractionation
Fractionation of mitochondria isolated from rat liver or HEK293 cells into inner membrane, outer membrane, and matrix components was performed exactly as previously described (Hoppel et al., 1998). Equal amounts of protein were loaded on SDS gels for antiBcl-x immunoblot analysis.
Sodium carbonate extractions
Crude mitochondria or microsomes (as prepared above) were resuspended in 0.1 M sodium carbonate (pH 12) and incubated for 20 min on ice. After centrifugation, the supernatant (containing the alkali-extractable proteins) was titrated to neutral pH with HCl. The pellet (containing the alkali-resistant fraction) was washed three times and then resuspended in buffer H (see above) containing 1% SDS. About 10% of the crude mitochondria or microsomal fraction was directly solubilized in buffer H containing 1% SDS (detergent control).
SDS-PAGE and Western blotting
30 µg of protein from subcellular fractions and sodium carbonate extractions were run on 15% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane (BDH), and immunodetected by anti-h/m/rBcl-x (S-18, 1:2,000; Santa Cruz Biotechnology, Inc.), antihBcl-2 (clone 124, 1:1,000; DakoCytomation), antimBcl-2 (10C4, 1:1000; Zymed Laboratories), anti-GFP/JL-8 (Living colors®, 1:1,000; Invitrogen), anti-KDEL (1:1,000; StressGen Biotechnologies), anti-grp78/Bip (1:1,000; StressGen Biotechnologies), and anti-COX/VIc (1:300; Molecular Probes) primary antibodies followed by peroxidase-coupled, Fc-specific goat antirabbit or antimouse secondary antibodies (Sigma-Aldrich). Immunodetection was performed by ECL (Pierce Chemical Co.). Equal protein loading was confirmed by amido black staining of the membrane.
Immunofluorescence analysis
Rat embryo fibroblasts (R6) or human epithelioid cervical carcinoma (HeLa) cells were grown on 12-mm glass coverslips and then were transiently transfected with 0.8 µg of plasmid DNA and 3.2 µl of Superfect as described above. At 24 h after transfection, cells were fixed in 4% paraformaldehyde and permeabilized with 0.05% saponin and ice cold acetone. The cells were incubated with anti-h/m/rBcl-xL (S-18, 1:150; Santa Cruz Biotechnology, Inc.), antihBcl-2 (clone 124, 1:100, DakoCytomation), or antimBcl-2 (10C4, 1:100; Zymed Laboratories) in the presence of either anticytochrome c (1:50; BD Biosciences), antiTOM-20 (1:300; gift from B. Hanson, Institute of Molecular and Cell Biology, Singapore), or anti-calnexin (1:100; StressGen Biotechnologies) as colocalization markers for 90 min followed by fluorescein- and/or Texas redconjugated goat antirabbit or antimouse secondary antibodies (Jackson ImmunoResearch Laboratories) for another 60 min. After postfixation in 4% paraformaldehyde containing 2 µg/ml Hoechst 33342 dye (Molecular Probes), the anti-fading agent Slowfade (Molecular Probes) was added, and the cells were viewed under a ZEISS Axioplan fluorescence microscope using standard filter for green and red fluorescence. Pictures were taken with a ZEISS digital camera and processed with the ZEISS AxioVision 3.06 software.
In vitro transcriptiontranslation (IVTT)
The TNT Quick T7-coupled reticulocyte lysate system (Promega) was used essentially as described by the manufacturer. In vitro transcriptiontranslation (IVTT) was performed at 30°C for 60 min in the presence of 0.6 mCi/ml [35S]methionine (Amersham Biosciences) in a volume of 25 µl. The reaction was stopped by the addition of 10 µg/ml cycloheximide (CHX). The lysate was centrifuged for 15 min at 12,000 g to remove possible nonsoluble aggregates. For membrane associationinsertion assays, freshly prepared crude mitochondria and microsomes from HEK293 cells were resuspended in MSH buffer (pH 7.5). 10 µl of the precleared IVTT product was mixed with 25 µl of membranes and incubated at 30°C for 20 min. After a centrifugation step (12,000 g for mitochondria, 100,000 g for microsomes), supernatants were collected and the membranes were washed three times in MSH buffer before analysis by SDS-PAGE and detection by autoradiography. For alkaline extraction of mitochondria/microsomes carrying IVVT products, the membranes were treated with sodium carbonate as described above.
Online supplemental material
Details about the generation of the various Bcl-2, Bcl-xL, and EGFP mutants by PCR can be found a http://www.jcb.org/cgi/content/full/jcb.200210084/DC1.
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Footnotes |
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* Abbreviations used in this paper: IVTT, in vitro transcriptiontranslation; MOM, mitochondrial outer membrane; TM, transmembrane.
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Acknowledgments |
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This work was supported by a grant from the Swiss National Science Foundation (31-57236.99) and the German-Israeli Foundation.
Submitted: 15 October 2002
Revised: 26 November 2002
Accepted: 27 November 2002
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References |
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