Mitochondrion-dependent N-terminal Processing of Outer Membrane Mcl-1 Protein Removes an Essential Mule/Lasu1 Protein-binding Site*

Mcl-1, a pro-survival member of the Bcl-2 family located at the mitochondrial outer membrane, is subject to constitutive ubiquitylation by the Bcl-2 homology 3-only E3 ligase, Mule/Lasu1, resulting in rapid steady-state degradation via the proteasome. Insertion of newly synthesized Mcl-1 into the mitochondrial outer membrane is dependent on its C-terminal transmembrane segment, but once inserted, the N terminus of a portion of the Mcl-1 molecules can be subject to proteolytic processing. Remarkably, this processing requires an intact electrochemical potential across the inner membrane. Three lines of evidence directed at the endogenous protein, however, indicate that the resulting Mcl-1ΔN isoform resides in the outer membrane: (i) full-length Mcl-1 and Mcl-1ΔN resist extraction by alkali but are accessible to exogenous protease; (ii) almost the entire populations of Mcl-1 and Mcl-1ΔN are accessible to the membrane-impermeant Cys-reactive agent 4-acetamido-4′-[(iodoacetyl)amino]stilbene-2,2′-disulfonic acid; and (iii) Mcl-1 and Mcl-1ΔN exhibit equivalent chemical cross-linking to Bak in intact mitochondria, an Mcl-1 binding partner located in the outer membrane. In addition to the Mule Bcl-2 homology 3 domain, we show that interaction between Mcl-1 and Mule also requires the extreme N terminus of Mcl-1, which is lacking in Mcl-1ΔN. Thus, Mcl-1ΔN does not interact with Mule, exhibits reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.

reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.
A plethora of cell-intrinsic pathways can activate apoptosis, commensurate with the diverse roles that programmed cell death plays in development, immunity, and disease. Many of these pathways are both executed and regulated by the Bcl-2 family of proteins. The family is comprised of three groups, defined according to their function and content of Bcl-2 homology (BH) 9 domains (1-3): the pro-apoptotic effectors, including Bax and Bak, contain BH1, BH2, and BH3 domains, as well as a C-terminal transmembrane segment that selectively targets these proteins to the membranes of mitochondria and endoplasmic reticulum; the pro-survival members Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, and Bcl-A1 have the same overall architecture as Bax and Bak but in addition contain a BH4 domain located toward their N termini; and finally, a large and diverse group within this family contains only a single BH3 domain. The latter, the BH3-only, proteins couple upstream death stimuli to downstream regulation of the multiple BH domain members. Considerable work has begun to provide an understanding of the regulation and function of specific Bcl-2 family members, which has helped to elucidate a number of important cell death pathways.
Mcl-1 was discovered based on its increased expression during cell commitment to differentiation in a human myeloid leukemia cell line (4). It has since been shown to be essential in the early stages of embryogenesis as Mcl-1 knock-out mice display peri-implantation lethality (5). Furthermore, it is necessary for the development and maintenance of B and T lymphocytes and for the survival of hematopoietic stem cells (6,7). Similar to other pro-survival Bcl-2 family members, elevated levels of Mcl-1 have also been shown to play a critical role in the initiation and progression of certain cancers. In particular, Mcl-1 is overexpressed in a number of human hematological cancers, including B cell lymphoma and chronic lymphocytic leukemia, as well as in solid tumor malignancies such as hepatocellular, pancreatic, and cervical carcinomas (8 -12).
Mcl-1 is uniquely subject to extensive regulation (13)(14)(15). This includes the regulation of rapid Mcl-1 protein turnover (16,17), in which degradation by the 26 S proteasome system is mediated by at least three E3 ubiquitin ligases: Mule/Lasu1, a BH3-only protein that selectively binds Mcl-1 among Bcl-2 family members and contributes to constitutive Mcl-1 turnover (18,19); ␤TrCP, which controls Mcl-1 ubiquitylation and degradation in response to Mcl-1 phosphorylation by GSK-3␤ (20,21); and the tumor suppressor SCF(FBW7), which is lost in a number of cancers (22). Additionally, there may be other ubiquitylation pathways, as well as ubiquitin-independent mechanisms for degrading Mcl-1 (23). As mentioned above, there is selective pressure across many cancer indications to increase Mcl-1 expression (24), and this includes enhanced Mcl-1 stabilization because of elevated expression of the de-ubiquitinase USP9X (25). Alternatively, N-terminal truncation of Mcl-1 is associated with stabilization and abundant expression in many tumor cells (26), but the underlying mechanisms remain to be determined.
Here, we show that following insertion of Mcl-1 into the mitochondrial outer membrane, the N terminus of Mcl-1 can be processed by a mechanism that depends on an intact electrochemical potential across the inner membrane. We show that although both isoforms localize to the outer mitochondrial membrane and inhibit apoptosis following cellular stress, the N-terminally truncated isoform evades interaction with Mule/ Lasu1, exhibits reduced ubiquitylation, and resists the hyperrapid degradation associated with full-length Mcl-1. As a result, Mcl-1⌬N retains its capacity to resist cell death in response to stresses such as UV irradiation that inhibit global protein synthesis.
Vector Construction-Full-length and N-terminally truncated mMcl-1 and hMcl-1 cDNAs were generated by PCR and cloned into pcDNA3. Mouse Mcl-1 G30E (GGG3 GAG) was obtained by random mutation. For Biacore binding studies, Mcl-1⌬TM(1-327) was generated by PCR and cloned into a pET29b vector modified to encode an N-terminal FLAG epitope along with the existing C-terminal polyhistidine (His 6 ) tag for purification. Murine stem cell virus-based vectors were introduced by retroviral infections as described previously by utilizing the Phoenix packaging system (27).
Treatments-For studies with UV treatment, cells were plated at a density of 3 ϫ 10 5 cells per well in 6-well dishes. The following day, media were removed, and cells were washed twice in cold PBS and then treated with the indicated dose of UV light using a UV Stratalinker 2400 (Stratagene); the media were then replaced, and cells were analyzed at the indicated time points. Unless indicated otherwise, cycloheximide was used at 100 g/ml in distilled H 2 O, and MG-132 was added at a concentration of 10 M.
Viability Measurements-Following treatment, floating cells were collected and pooled with trypsinized adherent cells. The cells were then resuspended in 500 l of staining buffer (PBS containing 2% FBS) containing 10 l of PI (50 g/ml). Cells were analyzed on a FACScan instrument (BD Biosciences). Matching blank control surfaces for each peptide were created as above except for the omission of the manual peptide injection. For binding analysis, recombinant FLAG-Mcl-1⌬TM-His 6 was simultaneously injected over the sample and blank control surfaces at the concentrations indicated. Sensograms derived from each concentration series were double referenced, and the K D value was determined by global analysis fit to a 1:1 Langmuir binding model using BIAEvaluation 3.2 software. Three independent runs were used to obtain the average K D value and standard deviation.
Transient Transfection and Immunoprecipitations-For transfections, HeLa and NIH3T3 cells were plated to 20% confluency in growth media. Cells were transfected by Lipofectamine TM (Invitrogen) 24 h later according to the manufacturer's protocol. For immunoprecipitations, cells were washed twice in PBS 48 h later and lysed at 4°C for 20 min in 1 ml of lysis buffer (50 mM HEPES, pH 7.7, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton X-100, 1 g/ml aprotinin, 1 g/l leupeptin, 1 g/l pepstatin, and 1 mM PMSF). The cell lysate was centrifuged at 12,000 rpm to remove membranes and insoluble debris. The cell lysate was pre-cleared with protein G-Sepharose beads (Amersham Biosciences), and the cleared cell lysate was incubated for 4 h at 4°C with the indicated antibody. Protein G-Sepharose was then added and incubated for 1.5 h. After incubation, the samples were pelleted and washed three times with lysis buffer and boiled in SDS sample buffer. After the samples were resolved by SDS-PAGE and transferred to nitrocellulose, immunoblotting was performed by standard procedures with the indicated antibodies, and blots were visualized with enhanced chemiluminescence. For densitometric analysis, ImageJ software (National Institutes of Health) was used. Following background subtraction, immunoprecipitation band intensities were divided by the corresponding lysate band intensities.
Biotin Pulldowns-HeLa cells were collected and lysed as described above and precleared for 1 h with streptavidin beads. Biotinylated synthetic peptide corresponding to the first 30 aa of mMcl-1 (Biotin-MFGLRRNAVIGLNLYCGGASLGAGGG-SPAG-NH 2 ) or a control biotinylated peptide (Biotin-PPRPRT-PGRPLSSYGMDSRPPMAIFEL-OH) were added at a concentration of 50 ng/mg of lysate and incubated for 3 h. Streptavidin beads were then added for 1 h, and samples were pelleted and washed as described above. Bound material was eluted by boiling in SDS-PAGE sample buffer.
Mcl-1 Pulse Labeling-MEFs were incubated in cysteine-free growth media containing twice-dialyzed FBS and then incubated for 15 min in the same fresh media. Media were then replaced with 4 ml of the same media containing 250 Ci/ml [ 35 S]methionine/cysteine labeling mixture. Cells were labeled for 45 min. Labeling was terminated with the addition of cycloheximide (100 g/ml) and harvested at the indicated times. Cells were washed three times in ice-cold PBS and lysed in 500 l of lysis buffer (50 mM HEPES, pH 7.7, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% Triton X-100, 1 g/ml aprotinin, 1 g/l leupeptin, 1 g/l pepstatin, and 1 mM PMSF). 500 g of protein from each lysate was pre-cleared with rabbit IgG antibody. The cleared lysates were then incubated with 2 g of anti-Mcl-1 antibody and recovered with protein A-Sepharose.
Mcl-1 was resolved on SDS-PAGE and transferred to a PVDF membrane. Radioactive signal was detected with autoradiographic film. The PVDF membrane was then immunoblotted with the indicated antibodies. Subcellular Fractionation-Cells (ϳ5 ϫ 10 7 cells) were collected and homogenized in 500 l of 200 mM mannitol, 70 mM sucrose, 10 mM HEPES, pH 7.5, 1 mM EGTA using a Teflon glass homogenizer maintained in ice water. The total homogenate was centrifuged in a microcentrifuge for 10 min at 4°C at 1800 rpm (600 ϫ g). The supernatant was designated S1 (containing mitochondria). S1 was centrifuged for 10 min at 4°C at 9000 rpm to yield a pellet (P9) enriched in mitochondria; the supernatant was designated S9. Mitochondria containing fraction P9 was treated with exogenous trypsin (0.125 g/ml) for 30 min at 4°C with the addition of trypsin inhibitor (1.25 g/ml) either at time 0 or after 30 min. Alkaline extraction was carried out by extracting P9 with 0.1 M Na 2 CO 3 , pH 11.5. Chemical labeling of the proteins in the P9 fraction with 4-acetamido-4Ј-[(iodoacetyl)amino]stilbene-2,2Ј-disulfonic acid (IASD) (Molecular Probes) was carried out exactly as described previously (28). Cross-linking of Mcl-1 and Bak using LC-succinimidyl 4(Nmaleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) was performed exactly as described previously (29) except that the post-nuclear S1 fraction was used.
Mitochondrial Protein Import-For an in vitro import reaction, mMcl-1 mRNA was in vitro translated (IVT) in rabbit reticulocyte lysate for 15 min at 30 0 after which time S1 or S9 was added at the ratio of IVT:S1/S9 of 1:4. The import reactions were carried out with the indicated fractions at 30°C for at least 6 h. When examined, 0.1 M CCCP or 5 M MG132 was added to S1 prior to the addition of IVT. Immunofluorescence

RESULTS AND DISCUSSION
To investigate Mcl-1 expression, we utilized SV40-transformed MEFs derived from WT and Mcl-1 Ϫ/Ϫ mice (30). High resolution gel electrophoresis and combined immunoprecipitation and immunoblot with an antibody that recognizes either the extreme N terminus or internal epitopes revealed the expression of both full-length Mcl-1 (ϳ40 kDa) and a truncated Mcl-1⌬N isoform (ϳ36 kDa) (Fig. 1, A and B). The latter roughly co-migrated with recombinant Mcl-1⌬N lacking aa 2-30, whereas the former co-migrated with the recombinant primary in vitro translation product of full-length Mcl-1 (Fig.  1C). In certain gels, an apparent intermediate size band was observed (e.g. see Fig. 2A and discussed below). Mcl-1 and Mcl-1⌬N were recovered in a heavy membrane fraction enriched in mitochondria (Fig. 1D) and exhibited equivalent co-localization with mitochondria as judged by immunofluorescence microscopy (Fig. 1E). Both isoforms co-immunoprecipitated with the established Mcl-1 outer membrane binding partner Bak (supplemental Fig. S1) (4,15), indicating that they both have functional BH3-binding modules. As expected (13), however, the two isoforms responded very differently to a blockade of global protein synthesis (conferred by the translation inhib-itor CHX or treatment with UV irradiation). As shown in Fig.  1F, full-length Mcl-1 but not Mcl-1⌬N rapidly disappeared in response to these treatments. Furthermore, the proteasome inhibitor MG132 inhibited this loss of full-length Mcl-1, resulting in its accumulation, but it had little effect on Mcl-1⌬N. Recombinant murine (supplemental Fig. S2) and human Mcl-1⌬N2-30 (supplemental Fig. S3) also exhibited relative stability in transfected cells following treatment with CHX or UV light. Importantly, transfected recombinant Mcl-1⌬N2-30 retained the ability to confer resistance to cell death in response to irradiation to an extent similar to that observed for full-length Mcl-1 (Fig. 1G).  JULY 15, 2011 • VOLUME 286 • NUMBER 28

Mcl-1 and Mcl-1⌬N Are Located in the Mitochondrial Outer
Membrane-A recent study based on siRNA knockdown suggested that the N terminus of Mcl-1 is subject to mitochondrial processing by the matrix-localized processing peptidase and concluded that this was due to import of Mcl-1 into the interior of the organelle, leaving Mcl-1⌬N within an internal compartment (31). In contrast, we find that Mcl-1⌬N is anchored in the outer membrane and exposed to the cytoplasm, consistent with the earlier findings that the Mcl-1 C-terminal transmembrane (TM) segment is both necessary and sufficient to target and insert Mcl-1 into the outer membrane lipid bilayer (32). Thus, removal of the N-terminal 2-30 aa of Mcl-1 did not interfere with the ability of ectopic expressed Mcl-1⌬N2-30 to target and localize to mitochondria in transfected cells (Fig. 1E). In isolated intact mitochondria, endogenous Mcl-1 and Mcl-1⌬N were both found to be resistant to alkaline extraction, pH 11.5, indicative of membrane integration, and both were sensitive to exogenous trypsin, indicating that they are surface-exposed; in contrast, cytochrome c, an intermembrane space protein, was extracted by alkali and resisted trypsin degradation ( Fig. 2A). To extend this analysis, we probed Mcl-1 and Mcl-1⌬N in isolated mitochondria with the thiol-conjugating agent IASD, which cannot enter or cross membranes and has previously been used to deduce the topology of surface versus membraneintegrated Cys-containing domains of Bcl-2 in the mitochondrial outer membrane (28). Conjugation to surface-exposed Cys residues could be conveniently assayed because it resulted in a mobility shift in Bcl-2 following gel electrophoresis (28). Similarly, exposure of isolated intact mitochondria to IASD caused a quantitative mobility shift of both Mcl-1 (contains 2 Cys) and Mcl-1⌬N (contains one Cys) following gel electrophoresis (Fig. 2B), consistent with a location of Mcl-1 and Mcl-1⌬N in the outer membrane. Of note, for this assay, cells had been briefly pulsed with CHX to enrich for Mcl-1⌬N prior to the isolation of mitochondria. A similar quantitative shift following IASD treatment was observed for Bak (Fig. 2B). In contrast, the intra-mitochondrial protein manganese superoxide dismutase exhibited a mobility shift in response to IASD only if the mem- branes were disrupted either by detergent or sonication, whereas outer membrane Bak was unaffected by detergent (Fig.  2C). Thus, the two known forms of Mcl-1 and Bak appear to be primarily associated to the outer membrane of the organelle. Therefore, as expected, endogenous Mcl-1 and Mcl-1⌬N were both observed to physically interact with Bak under conditions where the outer membrane remains intact, based on chemical cross-linking with LC-succinimidyl 4(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) in intact mitochondria (Fig. 2D) (29). In this experiment, one sample of cells was pulsed with CHX prior to isolation of mitochondria to generate mitochondria primarily containing Mcl-1⌬N (Fig. 2D, top left  panel, 2nd lane, and bottom left panel, 4th lane). As seen in Fig.  2D (right panel, lane 6), this sample containing primarily Mcl-1⌬N clearly exhibited cross-linking to Bak. Collectively, therefore, the evidence indicates that endogenous Mcl-1⌬N is functional and is located in the mitochondrial outer membrane.

Processing of Mcl-1 Occurs at the Mitochondrial Outer Membrane and Requires an Intact Mitochondrial ⌬-Transfection
of cDNA encoding full-length Mcl-1 into Mcl-1-null MEFs generated the same isoform pattern as expressed in WT MEFs (Fig. 3A), indicating that these isoforms are generated from the same mRNA. However, whereas the possibility exists for translation to initiate at noncanonical translation start sites within codons at the N terminus (15), extensive mutagenesis excluded this possibility (data not shown). Remarkably, however, a single point mutation at codon 30, in which a glycine is changed to glutamic acid (G30E), strongly decreased the generation of the , and together with mMcl-1, IVT was analyzed by immunoblot with anti-mMcl-1 directed toward a C-terminal region of the protein. The blot was stripped and re-probed with anti-mMcl-1 specific for the mMcl-1 N terminus. D, WT MEFs were radiolabeled with 35 S-Met/Cys for 45 min and then chased by the addition of CHX. Mcl-1 was immunoprecipitated (IP) and resolved by SDS-PAGE and transferred to PVDF. 35 S-Mcl-1 was detected with autoradiography for 24 h, and subsequently total mMcl-1 was detected by immunoblotting. The arrowheads depict full-length and processed Mcl-1. E, mMcl-1 IVT product was incubated with S1 or S9 fractions from Mcl-1-null MEFs under protein import conditions at the indicated temperatures. Import mixtures together with input IVT and WT MEF cell lysate were analyzed by immunoblot. F, as in E, except import was conducted in the presence or absence of 5 M MG132 or 0.1 M CCCP. G, as in F, except that following import, mitochondria (mito) (P9) were recovered prior to analysis by immunoblot.
Mcl-1⌬N isoform (Fig. 3A), suggesting that processing requires either a sequence or a structural requirement present in Mcl-1. Importantly, ectopically expressed Mcl-1G30E co-localized with mitochondria (Fig. 3B), indicating that mitochondrial targeting and subsequent processing can be disconnected. This is consistent with the prior findings that the Mcl-1 TM segment is both necessary and sufficient for targeting Mcl-1 to the mitochondria (31). Moreover, ectopic expression of Mcl-1⌬TM (full-length Mcl-1 lacking the TM segment) in Mcl-1 Ϫ/Ϫ MEFs exhibited a complete lack of processing and therefore expression of Mcl-1⌬N(⌬TM) (Fig. 3C). We therefore conclude that processing of Mcl-1 to Mcl-1⌬N occurs primarily at the mitochondrial outer membrane.
Pulse labeling of 35 S-Mcl-1 followed by chase in the presence of CHX suggested a conversion of Mcl-1 to Mcl-1⌬N (Fig. 3D). Furthermore, analysis of the Mcl-1 N terminus by MITOPROT suggested a potential sequence similarity to mitochondrial inner membrane/matrix targeting signals (supplemental Fig.  S4) (30). Such sequences include positively charged amino acids and an enrichment of smaller size amino acids, with an intolerance for negatively charged amino acids (33). Interestingly, introduction of the negatively charged Glu at position 30, which interfered with processing (Fig. 3A), would also be expected to be incompatible with a matrix targeting function. Furthermore, siRNA-mediated knockdown of the matrix-localized pre-protein processing peptidase was shown to interfere with the generation of Mcl-1⌬N (31). Finally, processing was also shown to be dependent on mitochondrial ⌬, which is restricted to the inner membrane of the mitochondria. This was demonstrated by incubating the Mcl-1 primary translation product in rabbit reticulocyte lysate containing an S1 post-nuclear cell extract (contains intact mitochondria) or an S9 post-mitochondrial cell extract (lacks mitochondria) from Mcl-1 Ϫ/Ϫ MEFs under conditions of protein import (34). Mcl-1 processing was achieved by S1 at 37°C but not at 4°C, whereas the S9 fraction was inactive (Fig. 3E). Employing the uncoupler CCCP, which collapses mitochondrial inner membrane ⌬, processing of Mcl-1 was abolished (Fig. 3F). Unlike CCCP, the proteasome inhibitor MG132 had no effect on processing (Fig. 3F). The processed product generated by S1 remained with mitochondria and was subsequently recovered in the P9 mitochondrion-enriched pellet fraction (Fig. 3G). Of note, however, based on co-migration of import products with endogenous Mcl-1 and Mcl-1⌬N, the in vitro import assay appeared to generate the intermediate size product rather than the fully processed Mcl-1⌬N (Fig. 3, E and  G), despite significant alterations to the import reaction in an attempt to achieve faithful processing to the in vivo isoform.
There are at least two explanations to account for processing of Mcl-1 to Mcl-1⌬N at the mitochondrial outer membrane. Either the long flexible N-terminal region of Mcl-1 (35) can gain ⌬-dependent transient (i.e. reversible) access to the matrix processing machinery, although the protein is still anchored at the outer membrane, or processing at the outer membrane is dependent on a system that is sensitive to inner membrane ⌬.
Processing of Mcl-1 to Mcl-1⌬N Prevents Its Interaction with Mule/Lasu-Mule/Lasu1 appears to be responsible mainly for constitutive steady-state degradation of Mcl-1 (18,19); consequently, efficient siRNA knockdown of Mule results in increased levels of Mcl-1 (19). Like most proteins that interact with pro-survival members of the Bcl-2 family, Mule contains a BH3 domain (18,19). Notably, its BH3 module exhibits differential selectivity for binding to Mcl (18,19,36) were first analyzed by surface plasmon resonance. Interestingly, the Mule BH3 peptide exhibited the weakest binding affinity (65 nM) among this group (Fig. 4, A and B). However, it was also found that Mule interacted with full-length Mcl-1 but not with Mcl-1⌬N as revealed by co-immunoprecipitation (Fig.  4C). Deletion mutagenesis indicated that a region corresponding to aa 20 -30 may make an important contribution to this interaction (Fig. 4D).
To determine whether the N terminus of Mcl-1 can independently associate with Mule, we constructed a biotinylated peptide comprising the first 30 amino acids of Mcl-1 (N30). Endogenous Mule was recovered from HeLa cell extracts with streptavidin beads bound with biotin-N30 peptide but not with a biotinylated control peptide (Fig. 4E) (Fig. 5, top panel).
Concluding Remarks-The extent of Mcl-1 turnover at the mitochondrial outer membrane depends on the relative frequency of its interaction with proteins that govern its degradation. One such key regulator that appears responsible for the rapid constitutive degradation of Mcl-1 is the E3 ligase Mule/ Lasu1. Efficient degradation demands therefore that Mule can effectively compete with other Mcl-1 binding partners. Thus, for example, overexpression of the constitutive binding partner Bim results in elevated levels of full-length Mcl-1 (19). Here, we show that the interaction between Mcl-1 and Mule depends on the presence of the Mcl-1 N terminus, in addition to the Mule BH3 domain. Our results further demonstrate that Mcl-1 can be processed at its N terminus, thereby rendering Mcl-1⌬N unable to interact with Mule and resulting in relative stabilization of Mcl-1 compared with the hyper-rapid degradation that is characteristic of full-length Mcl-1. It remains to be determined if the N terminus of Mcl-1 likewise influences its interactions with other regulators of Mcl-1 stability. Several lines of evidence show that such processing occurs only after newly synthesized Mcl-1 reaches the mitochondrion and is dependent on its presence at the mitochondrion. Despite the fact that manipulations that interfere with inner membrane ⌬ inhibit Mcl-1 processing, both full-length and processed Mcl-1 appear to reside integrated via their TM at the outer membrane. Future studies will be required to determine the precise details associated with the mechanism of this processing and whether it is associated with reversible import of the Mcl-1 N terminus into the organelle or on ⌬-dependent events that indirectly influence processing activity at the mitochondrial surface. By whatever mechanism, however, a processing pathway has evolved to allow Mcl-1 to escape rapid steady-state loss in response to antagonists of global protein synthesis. Enhanced mitochondrial processing of Mcl-1 to its more stable Mcl-1⌬N isoform could represent a control point for Mcl-1 stabilization and thus a potential mechanism for a cell survival advantage in the face of stress stimuli that influence global protein synthesis.