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. 2012 Apr 29;14(6):575-83.
doi: 10.1038/ncb2488.

Anti-apoptotic MCL-1 Localizes to the Mitochondrial Matrix and Couples Mitochondrial Fusion to Respiration

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Free PMC article

Anti-apoptotic MCL-1 Localizes to the Mitochondrial Matrix and Couples Mitochondrial Fusion to Respiration

Rhonda M Perciavalle et al. Nat Cell Biol. .
Free PMC article

Abstract

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.

Figures

Figure 1
Figure 1. Deletion of Mcl-1 results in mitochondrial morphology defects
(a) Mcl-1f/f Rosa-ERCreT2 or wild-type (wt) Rosa-ERCreT2 MEFs (control MEFs) were treated with tamoxifen or DMSO for indicated times to induce deletion of endogenous Mcl-1 then immunoblotted for MCL-1 or Actin (loading control). (b) Mcl-1f/f Rosa-ERCreT2 or wt Rosa-ERCreT2 MEFS were treated with tamoxifen (48 hours), seeded, and counted by hemocytometer at the indicated times. The data represent averages and SEM of 3 independent experiments. (c) Mcl-1f/f Rosa-ERCreT2 or wt Rosa-ERCreT2 MEFs stably-expressing matrix-targeted DsRed-fluorescent protein were treated with tamoxifen or DMSO for 96 hours to induce deletion of endogenous Mcl-1, then imaged to detect mitochondrial morphology. Scale bars represent 10 μm. (d) Average shape factor of mitochondrial matrix for Mcl-1f/f Rosa-ERCreT2 or control MEFs 96 hours after deletion. A shape factor of 1.0 indicates circular mitochondria and the higher the number, the more reticular the network. Error bars represent the standard error of the mean (SEM) from 10 imaged cells (~3000 mitochondria) and statistical significance was determined by unpaired t-test (*p<0.01). (e) Transmission electron micrographs of wt or Mcl-1-deficient MEFs. Scale bars represent 500 nm. (f) Quantification of mitochondrial cristae morphology from wt or Mcl-1-deficient MEFs. Mitochondria were counted from ~100 individual cells and scored for normal (lamellar) or ballooned and swollen cristae (disorganized). Error bars indicated the SEM from 3 independent experiments. (g) Enzymatic assays for indicated electron transport chain components from mouse liver mitochondria prepared from Mcl-1f/f Mx1-Cre or wt Mx1-Cre (wt) mice 14 days after treatment with pI-pC. Three independent animals were assayed in triplicate (n=9) and error bars indicated the SEM. Statistical significance was determined by unpaired t-test (*p<0.01).
Figure 2
Figure 2. MCL-1 resides in different sub-mitochondrial localizations
(a) Crude or Percoll-purified mouse liver mitochondria were western blotted for expression of MCL-1, Protein Disulphide Isomerase (PDI, endoplasmic reticulum, ER), or cytochrome c (inner membrane space, IMS). (b) Mouse liver mitochondria (HM) were sub-fractionated into outer mitochondrial membrane or mitoplast and western blotted for MCL-1, BCL-XL (outer mitochondrial membrane, OMM), Prohibitin1 (inner mitochondrial membrane, IMM), or MnSOD (matrix). (c) Mouse liver mitochondria (HM) were swollen and sonicated to disrupt membranes, washed with alkali buffer (pH 11.5) to detach loosely-associated proteins from membranes, and then re-isolated by ultra-centrifugation. Supernatant (Supe) and membrane fractions (Pellet) were western blotted for MCL-1, Prohibitin1, or MnSOD. (d, e, & f) Mitochondria isolated from (d) wt MEFs, (e) wt mouse liver, or (f) human Non-Small Cell Lung Cancer cells were subjected to proteinase K (PK) proteolysis to digest exposed proteins, osmotic shock (OS) was used to disrupt the outer mitochondrial membrane, and detergent (SDS) was used to disrupt both inner and outer mitochondrial membranes. Lysates were resolved and subjected to immunoblot analyses. Submitochondrial markers used are BCL-2 (OMM), Opa1 (IMM/IMS), and MnSOD. (g) Mitochondria isolated from wt MEFs transfected with control or siRNA-oligos specific to TOM40 or TIM50 were subjected to protease treatment as described in d.
Figure 3
Figure 3. MCL-1 mutants restrict mitochondrial localization
(a) Schematic illustration of MCL-1 mutants. Yellow boxes indicate BCL-2 homology (BH) domains and the red box indicates the C-terminal hydrophobic domain. Point mutations are indicated with the original amino acid, site-position and mutated residue. N. crassa ATP-synthase MTS is indicated in green. (b) Confocal immunofluorescence of Mcl-1-deficient MEFs transiently-expressing indicated Mcl-1 constructs were fixed and stained for MCL-1 (green), mitochondria (Mitotracker, in red), and nucleus (DAPI, in blue). Co-localization of green and red channels is shown in yellow overlay. Size bars represent 10 μm. (c & e) Whole cell lysates (WCL), isolated heavy membranes (HM) enriched for mitochondria or cytosolic (cyto) sub-cellular fractions were isolated from Mcl-1-deleted MEFs stably-expressing indicated MCL-1 constructs. Fractions were western blotted for MCL-1, BAX (cytosolic marker) or MnSOD (mitochondrial marker). (d) Mitochondria from Mcl-1-deficient MEFs stably-expressing human BCL-2 (hBCL-2) or a chimeric fusion of the first 50 amino acids of MCL-1 to hBCL-2 (mMCL-150-hBCL-2) were subjected to protease treatment as described in Fig. 2d. Both the fusion protein and hBCL-2 were detected with anti-hBCL-2 antibody. (f) Mitochondria from Mcl-1-deficient MEFs stably-expressing indicated constructs were subjected to protease treatment as described in Fig. 2d.
Figure 4
Figure 4. Anti-apoptotic activity of MCL-1 requires localization to the outer mitochondrial membrane
(a) Mcl-1f/f Rosa-ERCreT2 MEFs stably-expressing indicated constructs were treated for 48 hours with tamoxifen to induce deletion of endogenous Mcl-1 then western blotted for MCL-1, hBCL-2, or Actin (loading control). Vector-expressing, tamoxifen-treated wt Rosa-ERCreT2 MEFs (lane 1) serve as control. (b & c) Rosa-ERCreT2 MEFs stably-expressing indicated constructs were treated for 96 hours with tamoxifen to induce deletion of endogenous Mcl-1 and were administered indicated doses of (b) staurosporine for 16 hours or (c) etoposide for 24 hours after which cell death was determined. Annexin-V and PI double-negative cells were scored as viable. Bars represent the average of 3 independent experiments and the error bars denote the SEM. Vector-expressing, tamoxifen-treated wt Rosa-ERCreT2 MEFs serve as control. (d) Lysates were immunoprecipitated with anti-BIM or anti-rat IgG antibody and immune complexes were resolved and immunoblotted for MCL-1 and hBCL-2. Endogenous murine BCL-2 (mBCL-2) serves as a control for equal BIM immunoprecipitation. 1/10th input of immunoprecipitation is depicted. Post-immunoprecipitated supernatants (Unbound Flow Through) indicate non-precipitated proteins remaining in lysate.
Figure 5
Figure 5. Lacking matrix-localized MCL-1 results in mitochondrial IMM structure and fusion defects
(a) Transmission electron micrographs (12,000X) of Mcl-1f/f Rosa-ERCreT2 MEFs stably-expressing indicated constructs were treated with tamoxifen to induce deletion of endogenous Mcl-l. Scale bars represent 500 nm. (b) Quantification of mitochondrial cristae morphology from Mcl-1f/f Rosa-ERCreT2 MEFs stably-expressing indicated constructs that were treated with tamoxifen to induce deletion of endogenous Mcl-1. Mitochondria were counted from ~100 individual cells and scored for normal (lamellar) or ballooned and swollen cristae (disorganized). Error bars indicated the SEM from 3 independent experiments. (c) Mitochondrial fusion was measured 96 hours after Mcl-1-deletion in Mcl-1f/f Rosa-ERCreT2 MEFs stably-expressing indicated constructs. Cells were transiently-transfected with photoactivatable-GFP (PA-GFP) targeted to the mitochondrial matrix. Depicted are representative images from 2, 30, and 60 minutes after PA-GFP activation. Scale bars represent 10 μm. (d) Quantitation of the rate of mitochondrial fusion measured 96 hours after Mcl-1-deletion as the ratio of remaining activated mitochondrial area. Data represent average and SEM of fluorescence area over time of 3 independent experiments (n=16 cells). As mitochondria fuse, the PA-GFP signal dilutes decreasing intensity of signal in the area of activation.
Figure 6
Figure 6. MCL-1 regulates mitochondrial bioenergetics
(a) Mcl-1f/f Rosa-ERCreT2 MEFs stably-expressing indicated constructs were grown in glucose-free media containing galactose and treated for 96 hours with tamoxifen and total cellular ATP was evaluated. Vector-expressing, tamoxifen-treated wt Rosa-ERCreT2 MEFs are positive control. Data represent the averages of 3 independent experiments and the error bars denote the SEM. Statistical significance was determined by unpaired t-test (*p<0.01). (b) Quantification of mitochondrial membrane potential from Mcl-1f/f Rosa-ERCreT2 MEFs, stably-expressing indicated constructs, were treated for 96 hours with tamoxifen and stained with 10 nM TMRM and imaged. Data represent average mean fluorescence intensities and SEM from 20 cells. (c & d) Oxygen consumption measured 96 hours after Mcl-1-deletion in Mcl-1f/f Rosa-ERCreT2 MEFs stably-expressing indicated constructs under (c) basal and (d) FCCP-uncoupled (maximal) respiration. Vector-expressing, tamoxifen-treated wt Rosa-ERCreT2 MEFs serve as positive control. Experiments were performed in triplicate and the bars represent averages (n=9) and the error bars denote the standard of deviation. Statistical significance was determined by unpaired t-test (*p<0.01, **p<0.05). (e) Mouse liver lysates from Mcl-1f/f Mx1-Cre (Mcl-1-deleted) or wt Mx1-Cre (wt) mice 14 days after treatment with pI-pC were lysed in RIPA and resolved by SDS-PAGE. Immunoblots were probed to determine expression of Complex I (NDUFA9), Complex II (Fp70), Complex III (Core 2), Complex IV (Cox I and Cox 2), ATP Synthase subunit F0 B (5F1), ATP Synthase F1 subunit beta (5B), Succinate dehydrogenase, MnSOD, and MCL-1. Asterix (*) denotes mtDNA-encoded protein (f) Assessment of mtDNA and nDNA by quantitative real-time PCR. Error bars represent the standard error of mean (n=3). (g & h) Mouse liver mitochondria from Mcl-1f/f Mx1-Cre (Mcl-1-deleted) or wt Mx1-Cre (wt) mice 14 days after treatment with pI-pC were lysed in digitonin and resolved by blue native gel polyacrylamide electrophoresis (BN-PAGE) and blotted to determine native complexes and supercomplexes (SC). (g) complex I (CI, detected by NDUFA9), complex II (CII, detected by Fp70), complex III (CIII, detected by Core 2) and complex IV (CIV, detected by Cox I) and (h) ATP Synthase subunit F0 B (5F1), F1 subunit beta (5B). The native migration of monomers, dimers, and oligomers are denoted.

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