HeLa cells were maintained as described (Pearce et al., 2007). Antibodies raised in rabbits were: anti-Mcl-1 #D35A5 (for immunoblot), anti-Bcl-xL #54H6, anti-Bax #2772, anti-Bcl-2 #50E3, anti-caspase-3 #9662, anti-pDrp1-616 #D9A1 and anti-pDrp1-637 #4867S (Cell Signaling Technology), anti-Bak #06-536 (Millipore), anti-IP3R1, anti-IP3R2 and anti-IP3R3 (for immunoprecipitation; IP) (Wojcikiewicz, 1995), anti-erlin2 (Pearce et al., 2007), and anti-Bok (Ke et al., 2012). Mouse monoclonal antibodies were: anti-Flag epitope (M2, Sigma), anti-IP3R3 #610313 (for immunoblot) and anti-Drp1 #611112 (BD Biosciences), anti-V5 epitope tag (GenScript), anti-Mcl-1 #RC13 (for IP) and anti-streptavidin #S10D4 (ThermoFisher), and anti-p97 (Research Diagnostics Inc.). Purified streptavidin was from BioLegend. PCR and Gibson reagents were from New England BioLabs. SDS-PAGE reagents were from Bio-Rad. Lipofectamine 2000 was from ThermoFisher. Cell culture dishes were from Corning. HRP-conjugated secondary antibodies and all other reagents not listed were from Sigma. Vectors encoding mouse and human Mcl-1 and Bok, and human Bak were kind gifts from Dr. T. Kaufmann (Echeverry et al., 2013). pCag-mouse BokWT (Schulman et al., 2016) and associated mutants used in Figure 4 (BokL34G and BokΔTM, which lacks amino acids 188-213) were created by PCR using existing primers (Schulman et al., 2013). BioRender was used to generate Figures 1A, ,2A,2A, ,3B3B and Supplementary Figures 2, 3.

TurboID-Bok construct characterization. (A) V5-TurboID-Bok fusion constructs, T-BokWT and T-BokL34G. (B) Immunoblot for biotin-labeled species (detected with streptavidin/anti-streptavidin) in lysates from Bok KO HeLa cells transfected as indicated to express T-BokWT or T-BokL34G, without or with 2 h media supplementation with 50 μM biotin. Immunoreactivity of T-Bok constructs was assessed with either anti-Bok or anti-V5 (middle and lowest panels, respectively). (C) Anti-IP3R1/IP3R3 IP (lanes 1–3) and lysates (either pre- or post-IP; lanes 4–9) from Bok KO HeLa cells transfected as indicated, probed in immunoblots for the proteins indicated; p97 serves as a loading control. Co-migrating IgG heavy chain seen in the Bok probe of IPs is indicated by the asterisk. A 53kDa background band seen in the Bok probe of Bok KO cell lysates (lane 4) is indicated by the plus sign. Because BokL34G is relatively unstable (Schulman et al., 2016), to obtain equal expression, the amount of cDNA transfected for T-BokL34G was double that for T-BokWT.

TurboID-BokWT and TurboID-BokL34G interactomes. (A) Localization of proteins identified by T-BokWT (purple) and T-BokL34G (red). Percentage values are the percent of proteins assigned to the specified subcellular compartment; as proteins were assigned to 1–3 compartments, percentages add to >100%. For further information, see Supplementary Tables 1, 2. (B) Comparison of proteins identified by T-BokWT and T-BokL34G. The proteins shown were present in at least 6/7 and 4/5 independent experiments, respectively.

TurboID-Bak and TurboID-Mcl-1 interactomes. (A) Immunoblot for biotin-labeled species (detected with streptavidin/anti-streptavidin) in lysates from Bok KO HeLa cells transfected as indicated to express T-Bak or T-Mcl-1, without or with 2 h media supplementation with 50 μM biotin. Immunoreactivity of TurboID constructs was assessed with either anti-Bak, anti-Mcl-1, or anti-V5 (2nd-4th panels, respectively). (B) Localization of proteins identified by T-Bak (blue) and T-Mcl-1 (green). Percentage values are the percent of proteins assigned to the specified subcellular compartment; as proteins were assigned to 1–3 compartments, percentages add to >100%. For further information, see Supplementary Tables 3, 4. (C) Comparison of proteins identified by T-BokWT, T-Bak, and T-Mcl-1. The proteins shown were present in at least 6/7, 2/3, and 2/3 independent experiments, respectively.

Overexpressed Mcl-1 and Bok interact with consequences on apoptotic signaling. (A) Bok KO HeLa cells were transfected to express 1F-mouse Mcl-1 and mouse BokWT and mutants as indicated for ∼18 h, and cell lysates and anti-Flag IPs were probed as indicated; p97 serves as a loading control. (B) Cleaved caspase-3 (cC3) immunoreactivity, visualized as an ∼17kDa band, was measured and quantified in Bok KO HeLa cells expressing 1F-mouse Mcl-1 and mouse BokWT and mutants as indicated for ∼18 h; a representative immunoblot is shown together with quantification of cC3 immunoreactivity using Image Lab software (mean ± SEM, n = 4). An unpaired t-test with Welch’s correction was used to determine significance; p < 0.005 is denoted by **, p < 0.0005 is denoted by ***, n.s. = not statistically significant. Data were graphed and analyzed using GraphPad Prism software. (C) Bok KO HeLa cells were transfected to express 1F-human Mcl-1 and human BokWT as indicated for ∼18 h, and cell lysates and anti-Flag IPs were probed as indicated; p97 serves as a loading control.

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