Mitochondrion-dependent N-terminal processing of outer membrane Mcl-1 protein removes an essential Mule/Lasu1 protein-binding site

Abstract

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.

FIGURE 1.

Differential steady-state stability of Mcl-1 and Mcl-1ΔN. A, Mcl-1^−/− and WT MEF whole cell lysates were analyzed by immunoblotting with the indicated antibodies. B, MEF cell lysates were subjected to immunoprecipitation (IP) with either α-mMcl-1 antibody or preimmune IgG, and the precipitates were analyzed by immunoblotting with α-mMcl-1 antibody against the C-terminal region. The immunoblot was then stripped and reprobed with α-mMcl-1(N-term). C, immunoblot of in vitro translated product of mMcl-1 and mMcl-1Δ2–30 and MEF cell lysate. White line denotes lanes from the same blot that are not shown. The lanes are from the same blot and the same exposure. D, MEF cell lysate was collected and fractionated into heavy membrane enriched in mitochondria, light membrane enriched in endoplasmic reticulum, and cytosol (Cyto) and subjected to immunoblot with the indicated antibodies. Tom20 and Bap31 are markers for mitochondria and endoplasmic reticulum, respectively. E, Mcl-1^−/− MEFs were transfected with vectors expressing mMcl-1 or mMcl-1ΔN30, and the expression of Mcl-1 proteins was visualized with immunofluorescence microscopy, as indicated. Endogenous cytochrome c was used as marker for mitochondria. White arrows indicate mitochondrial localization. F, MEFs were treated with either 100 μg/ml CHX or 20 mJ/cm² UV irradiation in the presence or absence of MG132 (100 μm) and analyzed by immunoblot at the indicated time points. G, hMcl-1ΔN2–30 confers resistance to UV-induced cell death. G, panel i, Tsc2^−/− MEFs were transduced with the indicated vectors. Cells were then UV-irradiated (20 mJ/cm²), and both expression and stability of exogenous hMcl-1 were analyzed 2 h later by immunoblotting. G, panel ii, Tsc2^−/− MEFs expressing constructs as in G, panel I, were treated with UV irradiation (16 h), and cell death was determined.

FIGURE 2.

Mcl-1ΔN isoform is present at the surface of the outer mitochondrial membrane. A, mitochondria isolated from MEFs were treated either with exogenous trypsin or extracted with Na₂CO₃, pH 11. 5, as described under “Experimental Procedures,” and analyzed by immunoblot with the indicated antibodies. Cyt c, cytochrome c. Mitos, mitochondria. B, as in A, except mitochondria were isolated following a 0- or 2-h pulse of MEFs with CHX to enrich for mMcl-1 ΔN, and the mitochondria were treated with IASD (see “Experimental Procedures”). C, as in B, except that untreated (UT) mitochondria were subjected to membrane disruption by sonication or 0.5% Triton prior to incubation with IASD, and probed with anti- manganese superoxide dismutase (MnSOD) (left panel) or anti-Bak (right panel). D, as in B, except mitochondria were isolated from MEFs following a 0- or 3.5-h pulse with CHX and were subjected to cross-linking with LC-succinimidyl 4(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate). After immunoprecipitation (IP) with anti-mMcl-1, cross-linked Bak-Mcl-1 adducts (denoted by black dots) were identified by immunoblot with anti-Bak (right panel). Upper left panel shows immunoblot of total cell lysate; lower left panel shows immunoblot of the anti-Mcl-1 immunoprecipitate probed with anti-Mcl-1. WB, Western blot.

FIGURE 3.

Mitochondrial processing of Mcl-1. A, Mcl-1^−/− MEFs were transfected with vector or vector expressing mMcl-1, mMcl-1ΔN30, or mMcl-1G30E, and together with cell extract from WT, MEFs were analyzed by immunoblot with mMcl-1 antibody. B, Mcl-1G30E transiently expressed in Mcl-1 null MEFs was analyzed by immunofluorescence microscopy in parallel with endogenous cytochrome c (Cyto c) White arrows indicate mitochondrial localization. C, Mcl-1^−/− MEFs were transfected with vector expressing mMcl-1ΔTM (aa 1–312), 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 ³⁵S-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. ³⁵S-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 μm MG132 or 0.1 μm CCCP. G, as in F, except that following import, mitochondria (mito) (P9) were recovered prior to analysis by immunoblot.

FIGURE 4.

N terminus of Mcl-1 promotes increased association with Mule. A and B, synthetic peptides corresponding to the BH3 domain of the indicated BH3-containing proteins were immobilized on Biacore CM-5 sensorchip surfaces via the N terminus of each peptide. Recombinant FLAG-Mcl-1ΔTM (Mcl-1 aa 1–347) was injected over the surface at the concentrations indicated. Sensorgrams 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 deviations. A representative sensorgram of immobilized BidBH3 is shown in A. RU, response units. C, mMcl-1 cDNA was transfected into HeLa cells, and whole cell lysates were immunoprecipitated (IP) with α-Mule antibody or preimmune IgG. Immunoprecipitates were immunoblotted with murine-specific mMcl-1 antibody. D, HeLa cells were transiently transfected with mMcl-1 lacking the N-terminal aa 2–10, 2–20, or 2–30, and cell lysates were immunoprecipitated with α-Mule antibody or preimmune IgG. To confirm a reduced interaction between Mcl-1Δ2–30 and Mule, densitometric analysis was performed with ImageJ software (National Institutes of Health). Values represent the immunoprecipitated band intensities (background subtracted) divided by the corresponding lysate band intensities. NS, nonspecific. E, HeLa cell lysates were precleared with streptavidin beads, incubated with biotin-N30 synthetic peptide corresponding to the first 30 aa of mMcl-1 or a control biotinylated synthetic peptide at a concentration of 50 ng/mg lysate protein, and biotin was precipitated using streptavidin beads.

FIGURE 5.

N terminus of Mcl-1 promotes ubiquitylation at steady state. HeLa cells were transfected with vectors expressing mouse Mcl-1 or mouse Mcl-1ΔN30. 48 h post-transfection, cells were treated with or without MG132 for 3 h. Cell lysates were prepared and subjected to immunoprecipitation (IP) using anti-mouse Mcl-1 antibody. Total cellular proteins or the immunoprecipitates were subjected to immunoblot with anti-ubiquitin or anti-mMcl-1 antibodies. Note: mini-gel electrophoresis was employed, accounting for the close migration of Mcl-1 and Mcl-1ΔN30. WB, Western blot.