Fig 1.
Interaction of BimEL with TOM complex components.
(A) Proteins identified as enriched in 3xHA-BimEL purifications compared to control cells from bax-/- bak-/- MEF cells. Columns display: Gene Names, fold enrichment (light labeling of 3xHA-BimEL against heavy labeling of untagged Bim (see S1 Table for details)). (B) Western blots showing results of co-IP using anti-HA antibodies in MEF bax-/- bak-/- cells overexpressing either untagged or 3xHA-tagged murine BimEL. Mitochondria enriched fractions were isolated followed by anti-HA-IP in the presence of 1% digitonin. Data are representative of 4 independent experiments (Tom70), 2 independent experiments (Tom40) or 3 independent experiments (Tom20). Levels of overexpressed untagged and tagged BimEL (Input) and HA-IP efficiency (compare IP and Unbound) is shown on the right. (C) Western blots showing results of anti-HA-IP in HeLa cells either overexpressing 3xHA-tagged murine BimEL (+tamoxifen (4HT), 100 nM for 24h) or not. To inhibit 3xHA-BimEL-induced cell death QVD (10μM) was added. Again the IP was done with mitochondria enriched fractions in the presence of 1% digitonin. Induction and IP of 3xHA-mBimEL is shown on the right. (D) BimEL/TOM20 interaction does not require binding to anti-apoptotic Bcl-2 family proteins. Western blots showing results of anti-HA-IP in HEK-293FT cells transient transfected for 24h with either untagged murine BimEL (neg. control), 3xHA-tagged BimEL (positive control) or BH3-mutant BimEL (3xHA-BimELΔΔ; unable to bind to antiapoptotic proteins [11]). Whole cell extracts (1% digitonin) were used for IP. To inhibit BimEL-induced cell death QVD (10μM) was added (n = 3).
Fig 2.
Analyses of BimEL levels at mitochondria after knock-down of TOM components.
Western blots showing the levels of endogenous BimEL (A, C) or overexpressed 3xHA-BimEL (B, D) in mitochondrial enriched fractions (mito) and cytosolic fractions isolated from HeLa cells. (A, B) Western blots of BimEL with or without doxycycline-induced single shRNA knock-down (KD) of Tom40- or Tom70 (+Dox, 1μg/ml, for 4d). (C, D) Western blots of BimEL after triple TOM receptor KD (shRNA Tom70, siRNA Tom22 and Tom20). shRNA directed against Tom70 was induced (+Dox, 1μg/ml) and one day later siRNA for Tom20 and Tom22 was added for additional 3 days before fractionation. Where 3xHA-BimEL was induced 10μM of QVD was added to inhibit cell death. Fractionation was done as described under Material and Methods. Solubilisation was done with 1% Triton X-100. Western blots of tubulin and mitochondrial Hsp60 serve as fractionation and loading controls (A-D) and CoxIV and NDUFA9 (subunits of the respiratory chain) are examples of proteins that depend on the TOM complex for mitochondrial import (C, D). The detailed experimental design for each experiment can be found in S2 Fig For A, C, D (n = 3), for B (n = 2). Compare also another experiment S1F Fig to Fig 2C and 2D.
Fig 3.
BimEL-induced cell death in the absence of TOM complex components.
(A) Apoptosis induction measured by the percentage of active caspase-3 positive HeLa cells. Expression of 3xHA-BimEL was induced (+tamoxifen (4HT), 100nM) 24h before measurement. Where indicated Tom40 or Tom70-specific shRNA was induced (+doxycycline, 1μg/ml) 4 days ahead of 3xHA-BimEL induction, and QVD (10μM) was added to some samples to inhibit apoptosis. Data show means/SEM of 4 independent experiments. The experimental design is shown on the right. (B) Apoptosis measured as the percentage of active caspase-3 positive HeLa cells. Expression of 3xHA-BimEL was induced (+tamoxifen (4HT), 100nM) 24h before measurement. Where indicated shRNA directed against Tom70 was induced (+doxycycline, 1μg/ml) 3 days ahead of 3xHA-BimEL induction and siRNA KD of Tom20 and Tom22 was performed 2 days ahead of 3xHA-BimEL induction. To inhibit apoptosis QVD (10μM) was also added to some samples. Data show means/SEM of 5 independent experiments. The experimental design is shown on the right.
Fig 4.
In vitro import of BimEL into the yeast OMM after trypsin digestion.
(A, B) Import of radiolabelled murine BimEL precursor protein into isolated wild-type yeast mitochondria after trypsin digestion of outer membrane receptors. Import was performed for 5min (A) or varyingly over the indicated period of times (B). Samples were subjected to carbonate extraction and analyzed via SDS-PAGE and digital autoradiography. (C) Quantification of import data for Fig 4B. Data show means/SEM of 3 independent experiments (see S1 dataset) (D) Western blots showing the efficiency of trypsin digestion of receptors of the OMM (OMM = outer mitochondrial membrane, IMS = inner membrane space). The OMM is still intact after the procedure, because Tom22, an OMM protein, is degraded and control proteins (Mcr1, an IMS marker protein and Aco1, a matrix marker protein) are protected from protease treatment.
Fig 5.
Dependency of BimEL import into yeast OMM and yeast cell death/growth on individual TOM components.
(A) Radiolabelled 35S-BimEL was imported over various time periods into mitochondria isolated from wild-type (WT) or tom20Δ yeast cells. Samples were subjected to carbonate extraction and the pellet fractions (containing membrane inserted proteins) were analyzed by SDS-PAGE and digital autoradiography. (B) Quantification of the 35S-BimEL import data from Fig 5A. Data show means/SEM of 3 independent experiments. (C) Yeast cell death/growth under Bax and Bim expression in WT, Tom40 KD or Tom70 KO strains on glycerol plates (serial dilution of yeast cells). Bim is constitutively expressed, Bax is expressed after removal of tetracycline.