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Vol. 17, Issue 9, 3952-3963, September 2006
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*Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan; Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei 110, Taiwan; and Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Submitted April 18, 2006;
Accepted June 26, 2006
Monitoring Editor: Janet Shaw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Several reports have demonstrated that enforced expression of Mcl-1 delays apoptotic cell death induced by various stimuli, including cytokine withdrawal, exposure to cytotoxic agents (etoposide, calcium ionophore, or UV irradiation), and viral infection (Zhou et al., 1997; Chao et al., 1998; Cuconati et al., 2003). Mcl-1 deficiency results in peri-implantation embryonic lethality (Rinkenberger et al., 2000). Conditional knockout mouse models further reveal that Mcl-1 is a critical regulator essential for development and maintenance of T and B lymphocytes, and for ensuring the homeostasis of early hematopoietic progenitors (Opferman et al., 2003, 2005). Current models suggest that Mcl-1 may modulate apoptotic cell death via direct interaction with the multidomain proapoptotic Bcl-2 family members, including Bax and Bak, and/or the BH3-only proteins, such as Bim, Puma, and Noxa (Chen et al., 2005; Kuwana et al., 2005; Willis et al., 2005).
The mitochondrion is one major subcellular compartment where the Bcl-2 family members reside and exert their biological functions. Bcl-2 constitutively associates with the outer membrane of mitochondria (MOM) as well as other extramitochondrial membranes (Krajewski et al., 1993; Nguyen et al., 1993; Lithgow et al., 1994; Kaufmann et al., 2003). Bcl-XL is found both in the cytosol and mitochondrial membranes in healthy cells (Hsu et al., 1997; Hausmann et al., 2000). On stimulation by the apoptotic stimuli, the cytosolic fraction of Bcl-XL translocates to mitochondria (Hsu et al., 1997; Nijhawan et al., 2003). Bax is normally cytosolic. But, in response to apoptotic signals, it undergoes a conformational change and translocates to the outer membrane of mitochondria (Hsu et al., 1997; Wolter et al., 1997; Hsu and Youle, 1998). Conversely, Bcl-w is active while it is loosely associated with mitochondria. During apoptosis, a BH3-only protein binds to its hydrophobic pockets and releases its C-terminal domain for membrane insertion (Wilson-Annan et al., 2003). Last, Mcl-1, which functions at an apical step in many apoptotic regulatory programs, is mainly localized to mitochondria, but it is also found to be present in other intracellular compartments, including nucleus and other extramitochondrial membranes (Liu et al., 2005; Yang et al., 1995). The localization of Mcl-1 to these various subcellular compartments seems to be regulated in a cell state–specific manner (Liu et al., 2005). Although the tail-anchoring property of the C-terminal hydrophobic domain (Nguyen et al., 1993; Kaufmann et al., 2003; Jeong et al., 2004) and the cis-elements involved in the inducible membrane translocation of some Bcl-2 family members have been extensively characterized (Hsu et al., 1997; Wolter et al., 1997; Hsu and Youle, 1998; Wilson-Annan et al., 2003), the cellular machinery with which this family of proteins is targeted into mitochondria has been much less explored.
Most mitochondrial proteins are encoded by nuclear genes and are synthesized as preproteins in cytosol and later imported into mitochondria via translocation complexes in the outer (TOM) and inner mitochondrial membranes. The TOM machinery consists of two import receptors, Tom20 and Tom70, and a number of other subunits that are arranged in a tightly bound complex termed the "general import pore" (Pfanner and Geissler, 2001; Endo and Kohda, 2002; Hoogenraad et al., 2002; Stojanovski et al., 2003). Tom20 binds preproteins with both N-terminal presequences as well as internal targeting signals, whereas Tom70 preferentially binds preproteins with internal targeting information (Brix et al., 1997). In this report, we present evidence that Mcl-1 interacts with Tom70 and that such interaction facilitates mitochondrial targeting of Mcl-1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) except that pBTM116-hMcl-1
C27, which encodes a LexA-hMcl-1C27 fusion protein was used as bait to screen a human lymphocyte cDNA library.
Expression Vectors
pCDNA3-HA-hMcl-1, pCMV-Flag-hMcl-1, pCDNA3-hBcl-2, pFLAG-hBid, pCDNA3-FLAG-hBimS, pCDNA3-hBak, and pCDNA3-mBax are mammalian expression vectors for expressing various human (h) or mouse (m) proteins as indicated in the construct names. The expression vectors encoding the hMcl-1C102 or hMcl-1C165 mutants were generated by the insertion of a stop linker (5'-GGTAACTAATTAGGACC-3') into the XhoI or Asp718 site of the pCDNA3-HA-hMcl-1 vector. The expression vectors encoding all N-terminal truncation mutants of hMcl-1 (N90, N145, and N163) were generated by PCR by using specific forward primers and a common reverse primer, hM-M2, as indicated: hM-M2, 5'-CCCGAAGGTACCGAGAGA-3'; hMN90, 5'-GGATCCGCGACCCCCGCGAGG-3'; hMN145, 5'-GCGGATCCTTGGTCGGGGAATC-3'; and hMN163: 5'-GCGGATCCCCGCCGCCAGCAGA-3'.
The resultant PCR products were restricted with BamHI and KpnI before they were used to replace the corresponding fragment of the pCDNA3-HA-hMcl-1 vector. To construct expression vectors encoding the hMcl-1 mutants (L126A, D127A, and L126A/D127A), standard PCR-assisted mutagenesis-coupled cloning methods were carried out using hM-M2 in combination with the following primers (underlined nucleotides differ from the wild-type [wt] sequence): hM-L126A, 5'-GCCCGAAGAGGAGGCGGACGGGTACGAGC-3'; hM-D127A, 5'-CGAAGAGGAGCTGGCCGGGTACGAGCCG-3'; and hM-L126A/D127A, 5'-CCCGAAGAGGAGGCGGCCGGGTACGAGC-3'.
All constructs containing PCR derived regions were verified by DNA sequencing. pHA-hTom70 is a mammalian expression vector encoding an HA-tagged hTom70. This construct was derived by subcloning the hTom70 cDNA fragment from the pBluescript II SK(+)-KIAA0719 plasmid (a gift from Kazusa DNA Research Institute, Chiba, Japan) into the pcDNA3-HA vector. pGEX4T-Tom70 and pGEX4T-Tom70-R192A are bacterial expression vectors that direct the synthesis of wt or the clamp mutant (R192A) of Tom70 C-terminally fused to the glutathione S-transferase (GST) protein. pGST-hTom70N267 and pGST-hTom70N267 are bacterial expression vectors encoding GST-tagged hTom70 mutants containing only or without the N-terminal 267 amino acids, respectively.
Antibodies
Tom70-specific polyclonal antibodies were generated in guinea pigs (GP) by using bacterially produced histidine-tagged hTom70. Another Tom70-specific peptide antibody was raised in rabbits by using a synthetic peptide (QTEVAKKYGLKPPTL) corresponding to amino acid residues 584
598 of hTom70. Both antibodies were affinity purified using specific antigens cross-linked to CNBr-activated Sepharose (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Other antibodies used in this study include those specific to the hemagglutinin (HA)-tag (clone 12CA5; Roche Diagnostics, Indianapolis, IN); FLAG-tag (clone M2), 
-tubulin (both from Sigma-Aldrich, St. Louis, MO), Tom20 (FL-145), Mcl-1 (S-19), Bcl-2 (C21), Bax (N-20) (all from Santa Cruz Biotechnology, Santa Cruz, CA), Bak (Upstate Biotechnology, Lake Placid, NY), calnexin (clone C8.B6; Chemicon International, Temecula, CA), Hsp70 (clone C92F3A-5), and Hsp90 (clone AC88) (both from Calbiochem, San Diego, CA).
In Vitro GST Pull-Down Assays
The GST pull-down assay was carried out essentially as described previously (Liu et al., 2005) except that wt or various mutants of Mcl-1 transiently produced in CHOP cells were allowed to bind to bacterially produced GST, GST-hTom70, GST-hTom70N267, GST-hTom70N267, or GST-hTom70-R192A. The bound proteins were analyzed by immunoblotting using anti-HA or hMcl-1 antibody.
Indirect Immunofluorescence and Confocal Microscopy
293T cells, cultured on coverslips, were fixed with 4% (wt/vol) paraformaldehyde and 0.2% (vol/vol) glutaraldehyde in phosphate-buffered saline (PBS) for 20 min. After a brief wash with PBS, cells were blocked and permeabilized in PBS containing 0.5% normal goat serum and 0.2% saponin for another 20 min. The cells were then incubated with affinity-purified GP anti-Tom70 and rabbit anti-Mcl-1 antibody (S-19) in PBS containing 3% bovine serum albumin. After 1-h incubation at room temperature (RT), cells were washed with PBS and incubated for another 30 min at RT with Alexa Fluor 555-conjugated goat anti-GP IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA). After three washes with PBS, stained cells were counterstained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) and analyzed with confocal fluorescence microscopy (LSM 510 Meta; Carl Zeiss, Thornwood, NY). In some experiments, cells to be analyzed were first stained with MitoTracker before they were incubated with primary antibodies as described above.
Coimmunoprecipitation (co-IP) of Tom70 and hMcl-1
To analyze the interaction between endogenous Tom70 and hMcl-1, 293T cells were pretreated with 1 mM dithiobis(succinimidyl propionate) (Pierce Chemical, Rockford, IL) or vehicle control (dimethyl sulfoxide [DMSO]) for 30 min at RT before they were lysed in the SHE buffer (250 mM sucrose, 10 mM HEPES-KOH, pH7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate. Cell lysates were immunoprecipitated with control, GP anti-Tom70, or rabbit anti-hMcl-1 antibody. The immune complexes were then analyzed by immunoblotting by using antibodies specific to hMcl-1, Tom70, or Bcl-2. To analyze the interaction of ectopically overexpressed HA-Tom70 and FLAG-hMcl-1, a similar co-IP assay as described above was carried out except that cell lysates were from CHOP cells transiently producing these two tagged proteins (Liu et al., 2005) and appropriate control or tag antibodies were used in the subsequent immunoprecipitation and immunoblotting steps.
Subcellular Fractionation
Subcellular fractionation was carried out essentially as described by Nijhawan et al. (2003). The resultant three fractions, heavy membrane (HM), light membrane (LM), and cytosol, were analyzed by immunoblotting by using antibodies as indicated in the individual figures.
Opti-Prep Density Gradient Centrifugation
Cells to be analyzed were resuspended in ice-cold SHE buffer before they were broken up by passing through a 25-gauge needle 20 times. After two centrifugation steps at 1020 x g for 10 min to remove the nuclei and unbroken cells, the postnuclear lysates (PNLs) were layered on top of a 2.5–27.5% (wt/vol) linear iodixanol gradient (Opti-Prep; Axis-Shield, Oslo, Norway) in buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptine, and 10 µg/ml aprotinin) containing 250 mM sucrose. The gradients were then centrifuged at 200,000 x g for 3 h at 4°C in an SW41 rotor. Samples were sequentially collected from the top of the gradients into 20 0.5-ml fractions. The fractions were then analyzed by immunoblot by using antibodies specific to Mcl-1, Tom20 (mitochondria marker), calnexin (endoplasmic reticulum [ER] marker), and Bax (cytosolic marker).
In Vitro Mitochondrial Import Assay
Mitochondria were purified according to a published protocol (Schnaitman and Greenawalt, 1968) except that the C57BL/6J mouse liver was used as a source. The in vitro mitochondrial import assay was then carried out using freshly isolated mitochondria and 35S-labeled proteins (synthesized by the TNT-coupled reticulocyte lysate system; Promega, Madison, WI) essentially as described by Suzuki et al. (2002). The imported (pellet; P) and unimported fractions (supernatant; S) were subjected to SDS-PAGE, and specific signals were visualized by fluorography and quantified using a Bioimage Analyzer FLA5000 (Fuji, Tokyo, Japan). In some experiments, the imported fractions (P) were further extracted with 100 mM Na2CO3 (pH 11.5 or 10.8) for 30 min on ice and were then centrifuged at 100,000 x g for 30 min to separate the alkali-resistant (P) and -extractable (S) fractions. To examine the effect of Tom70 antibodies on the import reaction, mitochondria to be used in the import assay were preincubated with control or anti-Tom70 antibodies at 0°C for 30 min before the 35S-labeled proteins were added to the import system.
Generation of Stable Tom70-Knockdown K562 Cells
To knockdown Tom 70 expression in K562 cells, a Tom70 small interfering RNA (siRNA)-expressing vector (pSNG-siTom70) was constructed by inserting a pair of the Tom70 siRNA target sequence (5'-CCTCTGATGCCATCTCCAC-3') in a reverse orientation into the pSUPER-neo+gfp vector (OligoEngine, Seattle, WA) according to the manufacturer’s protocol. K562 cells stably transfected with the control or the pSNG-siTom70 vector were selected in growth medium supplemented with 1 mg/ml G418. Two independent clones (K-2 and K-11) with a best Tom70 knockdown efficiency and a pooled mixture of cells transfected with the control vector (Kv) were selected for further analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C27) as bait. By screening 9 x 106 clones from a human lymphocyte cDNA library, we obtained 58 positive clones. Sequence analysis revealed that these 58 clones represent cDNA fragments derived from 18 independent genes, some of which were Bcl-2 family members such as Bax, Bid, and Bik. In this study, we report the characterization of one of these 18 clones which turned out to be the human homologue of the fungal mitochondrial import receptor Tom70 (Alvarez-Dolado et al., 1999; Edmonson et al., 2002). Figure 1A illustrates four independent hTom70 cDNA clones that were fished out from our yeast two-hybrid screen.
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) was specifically pulled down by the GST-hTom70 fusion protein, but not by the GST protein alone. Next, we examined the in vivo interaction between ectopically overexpressed hMcl-1 and hTom70 by co-IP assays. In this assay, CHOP cells were transiently transfected with expressing vectors encoding HA-tagged hTom70 (HA-hTom70) and/or FLAG-tagged hMcl-1 (FLAG-hMcl-1). As shown in Figure 1C, FLAG-hMcl-1 specifically occurred in immune complexes precipitated by anti-HA antibody, but not by control IgG, and only under conditions when both FLAG-hMcl-1 and HA-hTom70 were coexpressed in the same transfected cells (Figure 1C, compare lanes 7–10). In a reciprocal experiment, the anti-FLAG antibody also specifically coprecipitated HA-hTom70 (Figure 1C, compare lanes 7 and 11–13). We next examined whether the interaction of Mcl-1 and Tom70 could be observed at endogenous levels of both proteins. For this experiment, a similar co-IP experiment (see Materials and Methods) was performed with cell lysates from human embryonic kidney cell line 293T. As shown in Figure 1D, although the GP anti-Tom70 antibody could specifically coprecipitate endogenous hMcl-1 (Figure 1D, compare lane 2 using control with lane 3 using Tom70 antibody), the amount of hMcl-1 present in the precipitated complex was very low (<1/200 of input proteins; compare lanes 1 and 3). However, under the same co-IP conditions, this Tom 70 antibody could precipitate much more (>50-fold) endogenous hMcl-1 from lysates prepared from 293T cells pretreated with a reversible cross-linking agent DSP (Figure 1D, compare lanes 1 and 3 and lanes 4 and 6). Under the latter conditions, endogenous Tom70 could also be detected in a reverse co-IP experiment using hMcl-1 antibody in the IP step (Figure 1D, lane 8). These results suggest that endogenous Tom70 and Mcl-1 interact with each other. However, their interaction may have been disrupted by the detergent used in the lysis buffer. Notably, under the same conditions, this GP anti-Tom70 antibody failed to coprecipitate Bcl-2, a protein known to target to MOM, from lysates prepared from 293T cells either with or without prior treatment with DSP (Figure 1D, lanes 3 and 6).
Mcl-1 Is Loosely Associated with the Outer Membrane of Mitochondria
Next, we compared the subcellular distributions of hTom70 and hMcl-1. Immunofluorescence analysis by using rabbit anti-Tom70 antibody and a mitochondrion-specific dye MitoTracker revealed that endogenous hTom70 colocalized with MitoTracker (Figure 2A, a–c). Interestingly, under our confocal microscopy settings, the Tom70 antibody gave a doughnut-shaped signal with MitoTracker staining in the center of this structure (see inset in Figure 2A, c). In contrast, in addition to being prominently localized to mitochondria, Mcl-1 is also present in other subcellular compartments (Figure 2A, d–f, and B). Subcellular fractionation analysis further confirmed that Tom70 is exclusively found in the mitochondria-enriched HM fraction, whereas Mcl-1 is predominantly found in the HM fraction with some minor populations residing in the LM and cytosolic fractions (Figure 2B, compare lanes 1–3). Immunoblotting using antibody that recognizes a protein marker specifically localized to ER (calnexin) or mitochondria (Tom20) indicated that the HM fraction analyzed in Figure 2B was slightly contaminated with the LM fraction. However, both the LM and the cytosolic fractions were free of any contaminated mitochondria. We also noticed that Mcl-1 and calnexin did not colocalize with each other in the immunostaining analysis (Supplemental Figure 1), albeit the detection of Mcl-1 in the LM fraction (Figure 2B). These results suggest that Mcl-1 is also localized to some extramitochondrial membranes that are distinct from those of the endoplasmic reticulum, a result that is consistent with that reported by Yang et al. (1995).
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58–60 kDa was released into the S fraction, a result that is very similar to that reported for the rat homologue of Tom70 (Suzuki et al., 2002). Furthermore, like Bcl-2 and Tom40, Tom70 was resistant to alkali extraction (see Materials and Methods) after it had integrated into mitochondria (Figure 2, D and E), suggesting that it is also stably integrated into MOM. In contrast, under the same conditions, a great amount of Mcl-1 (>50%) was sensitive to alkali extraction (Figure 2, D and E), indicating that Mcl-1 is loosely associated with MOM.
Mitochondrial Import of Mcl-1 Is Tom70 Dependent
Given that Tom70 is a known mitochondrial import receptor, we next examined whether Tom70 plays a role in the mitochondrial import of Mcl-1. We first isolated mitochondria from the mouse liver and performed an in vitro mitochondrial import assay to address this issue. Under our experimental conditions, cell-free translated Mcl-1 was imported into mitochondria with efficiency 50–90% (Figure 3A). Also, similar to the case with endogenous proteins (Figure 2, D and E), Mcl-1 imported into the isolated mitochondria was much sensitive to alkali treatment than Bcl-2 imported under the same conditions (Figure 3B). Furthermore, in this assay Bax and Bid, two Bcl-2 family members known to be localized to cytosol in the healthy cells, did not import into isolated mitochondria (Figure 3A, bottom two panels), and neither did the Mcl-1 mutant without the C-terminal transmembrane domain (Mcl-1C27; Figure 3A). Together, these results confirmed the specificity of our in vitro import system. Next, we examined whether an antibody that could block the interaction between Tom70 and Mcl-1 would prevent Mcl-1 from targeting to mitochondria in the aforementioned in vitro import system. For this experiment, we took advantage of our Tom70 antibody that was raised in rabbits using a peptide corresponding to the C-terminal end of Tom70. Unlike the GP anti-Tom70 antibody that could coimmunoprecipitate Mcl-1 (Figure 1D), the Tom70 peptide antibody raised in rabbits failed to do so (Supplemental Figure 2), suggesting that the latter antibody might interfere with the interaction between Mcl-1 and Tom70. As shown in Figure 4, the affinity-purified rabbit anti-Tom70 antibody, but not the control rabbit IgG, attenuated the in vitro import of Mcl-1 into mitochondria in a dose-dependent manner (Figure 4, A and C). Furthermore, consistent with the lack of interaction between Bcl-2 and Tom70 (Figure 1D), the same Tom70 neutralizing antibody did not exert any significant effect on the in vitro mitochondrial import of Bcl-2 (Figure 4, B and C).
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-tubulin (Figure 5A, middle) were not significantly altered. Interestingly, although the total protein levels of Mcl-1 are similar between the control and the Tom70 knockdown cells (Figure 5A), density gradient fractionation analysis revealed that the subcellular distributions of Mcl-1 in these two groups of cells are significantly different from each other (Figure 5, B and C). In control cells, Mcl-1 was distributed to cytosolic (C; fraction area 1–4), light membrane (L; fraction area 4–9), and mitochondrial (M; fraction area 9–15) fractions with an approximate ratio of 22:29:49 (Figure 5, B and C). However, in Tom70 knockdown cells, Mcl-1 was distributed to these three fractions with a ratio of 24:44:32 (C:L:M) (Figure 5, B and C). Under the same conditions, neither the subcellular distributions of Tom20 nor those of Bax were significantly affected. We could not examine whether knockdown of Tom70 would affect the subcellular distributions of Bcl-2 in K562 cells, because the endogenous Bcl-2 in these cells could not be efficiently detected by the Bcl-2 antibody used in this study. Together, these results suggest that Tom70 plays an important role in mitochondrial targeting of Mcl-1.
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C165 retaining 10–20% of that of the wt protein (see the legend to Figure 6A for definition of the Tom70-interacting activity). Alternatively, although deletion of N-terminal 90 amino acids as in the N90 mutant did not significantly affect Mcl-l’s binding to Tom70, further N-terminal deletion down to amino acid residue 145 or 163 nearly abolished Mcl-1’s ability to interact with Tom70 (Figure 6B, top, compare lanes 5 and 10; bottom, compare lanes 3 and 6). Although this experiment did not exclude the possibility that the C-terminal hydrophobic tail of Mcl-1 was involved in Mcl-1-Tom70 interaction, it did suggest that the region between amino acid residues 91 and 145 of hMcl-1 was required for such kind of protein–protein interaction. Within this region of Mcl-1, we noticed the presence of an EELD sequence, which is similar to the EEVD motif of two chaperone proteins (Hsp90 and Hsp70) (Figure 7A), that is recognized by two distinct tetratricopeptide repeat (TPR) domains (TPR1 and TPR2A) of the adaptor protein (p60/Hop) of these two chaperones (Scheufler et al., 2000; Brinker et al., 2002). Although the internal EELD sequence of Mcl-1 did not seem to conform to the rule revealed by the structural study where a TPR "clamp" domain and the EEVD motif interaction relies on bonds formed with the carboxylate group on the C-terminal aspartate residue (Scheufler et al., 2000), the following three findings still prompted us to examine whether the EELD motif is essential for Mcl-1 to interact with Tom70: 1) The N-terminal three TPR domains of human Tom70 are structurally similar to the TPR1 and TPR2A domains of p60/Hop (Young et al., 2003). 2) The Tom70 mutant containing the N-terminal three TPR domains as in GST-hTom70N267 bound to Mcl-1 with an affinity that was much higher than the mutant without this N-terminal region (Figure 7B). 3) The Bcl-2 member that does not interact with Tom70, e.g., Bcl-2 (Figure 1D), lacks such a characteristic EELD motif. We therefore generated Mcl-1 mutants with mutations in this motif. As shown in Figure 7C, mutation of either Leu-126 or Asp-127 to alanine markedly attenuated the interaction of Mcl-1 with Tom70 (compare lanes 7–9 in Figure 7C). Mutation of both residues to alanines (L126A and D127A) resulted into a mutant (DM) that lost more than 70% of Mcl-1’s binding affinity for Tom70 (Figure 7C, compare lanes 7 and 10; also see Supplemental Figure 3). The wt-like binding activity of the DM mutant to both Bim and Bak as determined by the co-IP assays (Figure 7D and 7E) suggested that the markedly attenuated interaction between the DM mutant of Mcl-1 and Tom70 was not due to a global conformational change of the DM mutant itself. Instead, this result suggests that the EELD motif of Mcl-1 resides in a domain that is critical for Mcl-1 to interact with Tom70.
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), could still interact with Mcl-1. To our surprise, under conditions when GST-Tom70 bound to Mcl-1 quite efficiently, the Mcl-1 binding activity of the GST-fused clamp mutant of Tom70 (GST-R192A) was markedly reduced (Figure 7F, compare lanes 2 and 3), a result that was very similar to the markedly compromised binding of GST-R192A to Hsp70 or to Hsp90 (Figure 7F, middle two panels). Together, our result suggests that the Mcl-1–Tom70 interaction is likely mediated via the internal EELD domain of Mcl-1 and the TPR clamp of the Tom70 molecule, albeit it does not seem to conform to the rule revealed by the structural studies as mentioned previously (Scheufler et al., 2000). In contrast, the Tom70 mutant study seems to be inconsistent with that obtained by the yeast-two hybrid screen where Tom70 clones without the N-terminal clamp domain were fished out by Mcl-1. Our best interpretation for these inconsistent results is that other structurally similar TPR motifs present in the core and/or the C-terminal region of Tom70 (Chan et al., 2006; Wu and Sha, 2006) may have mediated the Tom70–Mcl-1 interaction in the yeast two-hybrid assay, whose sensitivity apparently differs from that of the co-IP or the GST pull-down assays.
The DM Mutant Is Significantly Less Targeted to Mitochondria
Next, we examined whether the L126A and D127A mutations affect the subcellular localization of Mcl-1. To address this issue, we first established Ba/F3 derivatives stably overexpressing wt (clones W36 and W42) or the DM mutant (clones D54 and D71) of hMcl-1 (Figure 8A). Density gradient fractionation analysis was then used to compare the difference in subcellular distributions of these two proteins (one representative experiment is shown in Figure 8, B and C). Analysis of results from four independent experiments (Figure 8D) indicates that although the intracellular distribution ratios (C:L:M) of Mcl-1 (wt and DM) vary with the culture conditions of each independent experiment, under the same conditions, the DM mutant is consistently less targeted to mitochondria than the wt protein (12.3 ± 3.2%, n = 4, p = 0.018). Of note, in both cell clones (W36 and D71), the intracellular distributions of endogenous mMcl-1 were quite similar (Figure 8B), confirming the specificity of this assay. Next, we address the same issue by using another approach, i.e., compare the mitochondrial targeting efficiency of wt and the DM mutant by using the in vitro import assay. The result shown in Figure 9 indicates that the DM mutant was significantly less (compare 21–23% for the DM mutant vs. 43–49% for wt) targeted to mitochondria than the wt protein under assay conditions containing 0.01% NP-40, albeit no significant difference could be observed for these two proteins under our regular in vitro import assay, i.e., without the inclusion of NP-40 in the assay buffer. This latter result is probably due to the sensitivity limit of our assay system where a small difference in the targeting efficiency of two proteins can be differentiated only under a more stringent assay condition. Together, these two different assay methods further support the notion that mitochondrial targeting of Mcl-1 is mediated via a Tom70-dependent pathway.
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) and that it can potentially interact with other Bcl-2 family members, such as Bim and Bak (Supplemental Figure 4), the apoptotic response of the Tom70 knockdown cells may not truly reflect the functional aspect of the Tom70–Mcl-1 complex itself. More experiments are required to address this important issue.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C27) has a weaker Tom70 binding activity. Furthermore, this mutant cannot target to mitochondria, even though it contains the internal EELD domain. Our results suggest that the C-terminal hydrophobic tail and the internal EELD domain collaborate and direct mitochondrial targeting of Mcl-1.
Three isoforms of Mcl-1 have been noted in the immunoblotting analysis (Yang et al., 1995; Chao et al., 1998; Liu et al., 2005). In the Tom70 knockdown cells, we tend to see much less of the slowest migrating form, suggesting that the slowest migrating form is likely the form that is targeted to mitochondria and possibly via a Tom70-dependent import machinery as well. As mentioned, Mcl-1 is also localized to other extramitochondrial compartments. It remains to be determined what molecular mechanism(s) is responsible for sorting Mcl-1 into various subcellular localizations. Characterization of the identity and functions of various isoforms of Mcl-1 would certainly help address this intriguing question.
Mcl-1, Bcl-2, and Bcl-w are all found to be localized to mitochondria. However, their association with this organelle seems to be different. Bcl-2 is stably integrated into mitochondria, whereas Mcl-1 and Bcl-w are both loosely attached to this organelle. The machinery via which Bcl-w is imported into mitochondria has not been identified. Using the yeast system, the targeting of Bcl-2 into mitochondria was shown to be dependent on Tom20 but not on Tom70 (Motz et al., 2002). Furthermore, it was shown that such targeting requires the C-terminal hydrophobic tail of Bcl-2 (Motz et al., 2002). In contrast, Shirane and Nakayama (2003) reported that the mitochondrial FK506-binding protein 38 (FKBP38) interacts with Bcl-2 and plays an important role in targeting Bcl-2 to the mitochondrial membrane. An FKBP38 interacting domain was recently mapped to the unstructured loop of Bcl-2 (Kang et al., 2005). In this study, we demonstrate that mitochondrial targeting of Mcl-1 is facilitated by Tom70 via the internal EELD motif, which is apparently absent in the Bcl-2 molecule. Although we cannot exclude the possibility that the differential requirement of different import receptors for Bcl-2 and Mcl-1 is due to differences in the assay system (yeast vs. mammalian cells), we favor the interpretation that such differences in use of the import receptor may, at least partially, account for the different physical properties of Mcl-1 and Bcl-2 in association with mitochondria. This latter difference and other known differences in the tissue and subcellular distributions and affinities for various proapoptotic molecules (Chen et al., 2005) may all together account for the very different phenotypes of Mcl-1 and Bcl-2 knockout mice (Veis et al., 1993; Rinkenberger et al., 2000).
In the Tom70 knockdown cells, we observed that more Mcl-1 molecules than those in the control cells are redistributed to extramitochondrial fractions, albeit the exact subcellular location of these redistributed Mcl-1 molecules is not clear. This result suggests that under conditions when the positive mitochondrial import signal (mediated via the import receptor Tom70) is limiting, Mcl-1 is then randomly sorted to all intracellular locations. This interpretation is further supported by the results from experiment using Mcl-1 mutant (DM) with markedly reduced affinity for Tom70. In the latter study, the DM mutant is significantly less targeted to mitochondria than the wt protein. As mentioned, our results suggest that for optimal targeting to mitochondria the C-terminal hydrophobic and the internal EELD domains of Mcl-1 both need to collaborate with each other. This is in sharp contrast to that reported for Bcl-XL where the C-terminal transmembrane domain with some flanking basic residues is sufficient to target the linked reporter protein enhanced green fluorescent protein exclusively to mitochondria (Kaufmann et al., 2003). It was further proposed that lack of specific mitochondrial targeting ability of the C-terminal hydrophobic tail of both Mcl-1 and Bcl-2 is mainly due to lack of sufficient basicity in this region of protein sequences (Kaufmann et al., 2003). Alternatively, FKBP38 was shown to facilitate the mitochondrial import of Bcl-2 (Shirane and Nakayama, 2003) and likely did so via binding to the N-terminal unstructured loop of Bcl-2 (Kang et al., 2005). These results suggest that, like with Mcl-1, mitochondrial targeting of Bcl-2 requires the collaboration of two protein domains, one in the N terminus and the other in the C-terminal end. It remains to be determined whether requirement of two domains for mitochondrial targeting is a common property for those membrane proteins without a specific mitochondrial targeting signal in their membrane targeting/anchoring domain. Intriguingly, FKBP38 can also interact with Bcl-XL and facilitates its mitochondrial targeting (Shirane and Nakayama, 2003). It would be interesting to determine whether the FKBP38 binding domain resides in the C-terminal mitochondrial targeting sequence of Bcl-XL, because this region of protein alone was shown to be sufficient to confer on Bcl-XL a mitochondrial localization (Kaufmann et al., 2003).
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-04-0319) on July 5, 2006.
Address correspondence to: Hsin-Fang Yang-Yen (imbyy{at}gate.sinica.edu.tw)
Abbreviations used: co-IP, coimmunoprecipitation; GP, guinea pig; MOM, outer membrane of mitochondria.
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