Abstract
Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Ferriero DM. Neonatal brain injury. N. Engl J Med. 2004;351:1985–95.
Oliveira JB, Gupta S. Disorders of apoptosis: mechanisms for autoimmunity in primary immunodeficiency diseases. J Clin Immunol. 2008;28:20–8.
Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002;2:647–56.
Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49–63.
Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev. 2005;19:1294–305.
Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell. 2005;17:393–403.
Lee Y-J, Jeong S-Y, Karbowski M, Smith CL, Youle RJ. Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. Mol Biol Cell. 2004;15:5001–11.
Yu T, Fox RJ, Burwell LS, Yoon Y. Regulation of mitochondrial fission and apoptosis by the mitochondrial outer membrane protein hFis1. J Cell Sci. 2005;118:4141–51.
Weaver D, Eisner V, Liu X, Várnai P, Hunyady L, Gross A, et al. Distribution and apoptotic function of outer membrane proteins depend on mitochondrial fusion. Mol Cell. 2014;54:870–8.
Roy SS, Ehrlich AM, Craigen WJ, Hajnóczky G. VDAC2 is required for truncated BID‐induced mitochondrial apoptosis by recruiting BAK to the mitochondria. EMBO Rep. 2009;10:1341–7.
Naghdi S, Várnai P, Hajnóczky G. Motifs of VDAC2 required for mitochondrial Bak import and tBid-induced apoptosis. Proc Natl Acad Sci. 2015;112:E5590–E5599.
Yamagata H, Shimizu S, Nishida Y, Watanabe Y, Craigen W, Tsujimoto Y. Requirement of voltage-dependent anion channel 2 for pro-apoptotic activity of Bax. Oncogene 2009;28:3563–72.
Ma S, Nguyen T, Tan I, Ninnis R, Iyer S, Stroud D, et al. Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function. Cell Death Differ. 2014;21:1925–35.
Chin HS, Li MX, Tan IK, Ninnis RL, Reljic B, Scicluna K, et al. VDAC2 enables BAX to mediate apoptosis and limit tumor development. Nat Commun. 2018;9:4976.
Cheng EH-Y, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 2003;301:513–7.
O’Neill KL, Huang K, Zhang J, Chen Y, Luo X. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane. Genes Dev. 2016;30:973–88.
Djajawi TM, Liu L, Gong J-N, Huang AS, Luo M-J, Xu Z, et al. MARCH5 requires MTCH2 to coordinate proteasomal turnover of the MCL1: NOXA complex. Cell Death Differ. 2020;27:2484–99.
Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783.
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34:184–91.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339:819–23.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308.
Koike-Yusa H, Li Y, Tan E-P, Velasco-Herrera MDC, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol. 2014;32:267–73.
Aubrey BJ, Kelly GL, Kueh AJ, Brennan MS, O’Connor L, Milla L, et al. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo. Cell Rep. 2015;10:1422–32.
Waterhouse NJ, Steel R, Kluck R, Trapani JA, Assaying cytochrome C translocation during apoptosis. Signal Transduction Protocols. Springer, 2004, pp 307–13.
Johnston AJ, Hoogenraad J, Dougan DA, Truscott KN, Yano M, Mori M, et al. Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane. J Biol Chem. 2002;277:42197–204.
Hock DH, Reljic B, Ang C-S, Muellner-Wong L, Mountford HS, Compton AG, et al. HIGD2A is required for assembly of the COX3 module of human mitochondrial complex IV. Mol Cell Proteom. 2020;19:1145–60.
Eden E, Lipson D, Yogev S, Yakhini Z. Discovering motifs in ranked lists of DNA sequences. PLoS Comput Biol. 2007;3:e39.
König R, Chiang C-Y, Tu BP, Yan SF, DeJesus PD, Romero A, et al. A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods. 2007;4:847–9.
Wei MC, Zong W-X, Cheng EH-Y, Lindsten T, Panoutsakopoulou V, Ross AJ, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001;292:727–30.
Haraguchi M, Torii S, Matsuzawa S-I, Xie Z, Kitada S, Krajewski S, et al. Apoptotic protease activating factor 1 (Apaf-1)–independent cell death suppression by Bcl-2. J Exp Med. 2000;191:1709–20.
Tait SW, Green DR. Caspase-independent cell death: leaving the set without the final cut. Oncogene 2008;27:6452–61.
Gama V, Swahari V, Schafer J, Kole AJ, Evans A, Huang Y, et al. The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells. Sci Signal. 2014;7:ra67.
Tait SW, Parsons MJ, Llambi F, Bouchier-Hayes L, Connell S, Muñoz-Pinedo C, et al. Resistance to caspase-independent cell death requires persistence of intact mitochondria. Dev Cell. 2010;18:802–13.
Hostein I, Robertson D, DiStefano F, Workman P, Clarke PA. Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res. 2001;61:4003–9.
Subramanian A, Andronache A, Li YC, Wade M. Inhibition of MARCH5 ubiquitin ligase abrogates MCL1-dependent resistance to BH3 mimetics via NOXA. Oncotarget 2016;7:15986–6002.
Cherok E, Xu S, Li S, Das S, Meltzer WA, Zalzman M, et al. Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5–dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics. Mol Biol Cell. 2017;28:396–410.
Arai S, Varkaris A, Nouri M, Chen S, Xie L, Balk SP. MARCH5 mediates NOXA-dependent MCL1 degradation driven by kinase inhibitors and integrated stress response activation. Elife. 2020;9:e54954.
Haschka MD, Karbon G, Soratroi C, O’Neill KL, Luo X, Villunger A, MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment. Cell Death Differ. 2020;27:2297–312.
Karbowski M, Neutzner A, Youle RJ. The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division. J Cell Biol. 2007;178:71–84.
Park Y-Y, Lee S, Karbowski M, Neutzner A, Youle RJ, Cho H. Loss of MARCH5 mitochondrial E3 ubiquitin ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1. J Cell Sci. 2010;123:619–26.
Park Y-Y, Cho H, Mitofusin 1 is degraded at G 2/M phase through ubiquitylation by MARCH 5. Cell Div. 2012;7:25.
Yonashiro R, Ishido S, Kyo S, Fukuda T, Goto E, Matsuki Y, et al. A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J. 2006;25:3618–26.
Nakamura N, Kimura Y, Tokuda M, Honda S, Hirose S. MARCH‐V is a novel mitofusin 2‐and Drp1‐binding protein able to change mitochondrial morphology. EMBO Rep. 2006;7:1019–22.
Xu S, Cherok E, Das S, Li S, Roelofs BA, Ge SX, et al. Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein. Mol Biol Cell. 2016;27:349–59.
Sugiura A, Nagashima S, Tokuyama T, Amo T, Matsuki Y, Ishido S, et al. MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2. Mol Cell. 2013;51:20–34.
Phu L, Rose CM, Tea JS, Wall CE, Verschueren E, Cheung TK, et al. Dynamic regulation of mitochondrial import by the ubiquitin system. Mol Cell. 2020;77:1107–23.e1110.
Westphal D, Kluck R, Dewson G. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ. 2014;21:196–205.
Dewson G, Kratina T, Sim HW, Puthalakath H, Adams JM, Colman PM, et al. To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3: groove interactions. Mol Cell. 2008;30:369–80.
Griffiths GJ, Corfe BM, Savory P, Leech S, Degli Esposti M, Hickman JA, et al. Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak. Oncogene 2001;20:7668–76.
Brouwer JM, Westphal D, Dewson G, Robin AY, Uren RT, Bartolo R, et al. Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers. Mol Cell. 2014;55:938–46.
Alsop AE, Fennell SC, Bartolo RC, Tan IK, Dewson G, Kluck RM. Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis. Nat Commun. 2015;6:1–13.
Dewson G, Kratina T, Czabotar P, Day CL, Adams JM, Kluck RM. Bak activation for apoptosis involves oligomerization of dimers via their α6 helices. Mol Cell. 2009;36:696–703.
Iyer S, Bell F, Westphal D, Anwari K, Gulbis J, Smith B, et al. Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains. Cell Death Differ. 2015;22:1665.
Pang Y-P, Dai H, Smith A, Meng XW, Schneider PA, Kaufmann SH. Bak conformational changes induced by ligand binding: insight into BH3 domain binding and Bak homo-oligomerization. Sci Rep. 2012;2:257.
Li MX, Tan IK, Ma SB, Hockings C, Kratina T, Dengler MA, et al. BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove. Proc Natl Acad Sci. 2017;114:7629–34.
Uren RT, O’Hely M, Iyer S, Bartolo R, Shi MX, Brouwer JM, et al. Disordered clusters of Bak dimers rupture mitochondria during apoptosis. Elife 2017;6:e19944.
Bleicken S, Classen M, Padmavathi PV, Ishikawa T, Zeth K, Steinhoff H-J, et al. Molecular details of Bax activation, oligomerization, and membrane insertion. J Biol Chem. 2010;285:6636–47.
Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, et al. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nat Commun. 2015;6:1–11.
Llambi F, Moldoveanu T, Tait SW, Bouchier-Hayes L, Temirov J, McCormick LL, et al. A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol Cell. 2011;44:517–31.
Hockings C, Alsop AE, Fennell SC, Lee EF, Fairlie WD, Dewson G, et al. Mcl-1 and Bcl-x L sequestration of Bak confers differential resistance to BH3-only proteins. Cell Death Differ. 2018;25:721–34.
Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006;10:375–88.
Roberts AW, Stilgenbauer S, Seymour JF, Huang DC. Venetoclax in patients with previously treated chronic lymphocytic leukemia. Clin Cancer Res. 2017;23:4527–33.
Kotschy A, Szlavik Z, Murray J, Davidson J, Maragno AL, Le Toumelin-Braizat G, et al. The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature 2016;538:477–82.
Dewson G, Snowden R, Almond J, Dyer M, Cohen G. Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene 2003;22:2643–54.
Edlich F, Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C, et al. Bcl-x(L) Retrotranslocates Bax from the Mitochondria into the Cytosol. Cell 2011;145:104–16.
Lauterwasser J, Todt F, Zerbes RM, Nguyen TN, Craigen W, Lazarou M, et al. The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation. Sci Rep. 2016;6:1–11.
Todt F, Cakir Z, Reichenbach F, Emschermann F, Lauterwasser J, Kaiser A, et al. Differential retrotranslocation of mitochondrial Bax and Bak. EMBO J. 2015;34:67–80.
Jenner A, Pena-Blanco A, Salvador-Gallego R, Ugarte-Uribe B, Zollo C, Ganief T, et al. DRP1 interacts directly with BAX to induce its activation and apoptosis. EMBO J. 2022;41:e108587.
Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, et al. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell. 2008;14:193–204.
Bordt EA, Clerc P, Roelofs BA, Saladino AJ, Tretter L, Adam-Vizi V, et al. The Putative Drp1 Inhibitor mdivi-1 is a reversible mitochondrial complex I inhibitor that modulates reactive oxygen species. Dev Cell. 2017;40:583–94.e586
Bernardini JP, Brouwer JM, Tan IK, Sandow JJ, Huang S, Stafford CA, et al. Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy. EMBO J. 2019;38:e99916.
Zhong Q, Gao W, Du F, Wang X. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell 2005;121:1085–95.
Fang L, Hemion C, Goldblum D, Meyer P, Orgül S, Frank S, et al. Inactivation of MARCH5 prevents mitochondrial fragmentation and interferes with cell death in a neuronal cell model. PLoS One. 2012;7:e52637.
Wang J, Aung LH, Prabhakar BS, Li P, The mitochondrial ubiquitin ligase plays an anti‐apoptotic role in cardiomyocytes by regulating mitochondrial fission. J Cell Mol Med. 2016, 20: 2278–88.
van Delft MF, Chappaz S, Khakham Y, Bui CT, Debrincat MA, Lowes KN, et al. A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis. Nat Chem Biol. 2019;15:1057–66.
Aluvila S, Mandal T, Hustedt E, Fajer P, Choe JY, Oh KJ. Organization of the mitochondrial apoptotic BAK pore oligomerization of the bak homodimers. J Biol Chem. 2014;289:2537–51.
Cowan AD, Smith NA, Sandow JJ, Kapp EA, Rustam YH, Murphy JM, et al. BAK core dimers bind lipids and can be bridged by them. Nat Struct Mol Biol. 2020;27:1024–31.
Chipuk JE, McStay GP, Bharti A, Kuwana T, Clarke CJ, Siskind LJ, et al. Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis. Cell 2012;148:988–1000.
Vasquez-Montes V, Rodnin MV, Kyrychenko A, Ladokhin AS, Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL. Proc Natl Acad Sci. 2021;118:e2025834118.
Bond ST, Moody SC, Liu Y, Civelek M, Villanueva CJ, Gregorevic P, et al. Mitochondria dysfunction in aging and metabolic diseases: The E3 ligase MARCH5 is a PPARγ target gene that regulates mitochondria and metabolism in adipocytes. Am J Physiol-Endocrinol Metab. 2019;316:e293.
Kitakata H, Endo J, Matsushima H, Yamamoto S, Ikura H, Hirai A, et al. MITOL/MARCH5 determines the susceptibility of cardiomyocytes to doxorubicin-induced ferroptosis by regulating GSH homeostasis. J Mol Cell Cardiol. 2021;161:116–29.
Hosoi K-I, Miyata N, Mukai S, Furuki S, Okumoto K, Cheng EH, et al. The VDAC2–BAK axis regulates peroxisomal membrane permeability. J Cell Biol. 2017;216:709–22.
Deutsch EW, Bandeira N, Sharma V, Perez-Riverol Y, Carver JJ, Kundu DJ, et al. The ProteomeXchange consortium in 2020: enabling ‘big data’ approaches in proteomics. Nucleic Acids Res. 2020;48:D1145–D1152.
Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450.
Acknowledgements
We would like to thank Ruth Kluck, Peter Czabotar, Daniel Gray, and Andreas Strasser for helpful discussions and suggestions; Melissa Shi for technical assistance; Stephen Wilcox for DNA sequencing; Simon Cobbold for assistance with proteomic analysis; Prof Xu Luo for providing all BCL-2 genes knockout HCT116 cells; Christine White for technical support. We thank the Bio21 Mass Spectrometry and Proteomics Facility (MMSPF) for the provision of instrumentation, training, and technical support, and the Mito Foundation for the provision of instrumentation through the large equipment grant support scheme. This work was supported by scholarship fellowship and grants from the Australian National Health and Medical Research Council (NHMRC) to DAS (#1140851, #1140906); Melbourne University (MIRS and MIFRS scholarships to ASH and TMD); the Walter and Eliza Hall Institute of Medical Research (to ASH); the Bodhi Foundation and the McPhee Charitable Trust (to GD). Research was supported by an NHMRC Independent Research Institutes Infrastructure Support Scheme grant (361646) and Victorian State Government Operational Infrastructure Support grant.
Author information
Authors and Affiliations
Contributions
DCSH, MvD, and GD devised the study and interpreted data. ASH and HSC planned, performed experiments, and interpreted data. BR and DAS performed the proteomics and interpreted data. TMD, JNG, and IKLT generated reagents; ASH and MvD collated and analysed data. ASH, MvD, GD wrote the manuscript. All authors contributed to manuscript review.
Corresponding authors
Ethics declarations
Competing interests
GD, DCSH and MvD are employees of the Walter and Eliza Hall Institute which receives royalty payments relating to the use of Venclexta/venetoclax.
Ethics approval
This study did not include human participants or animal research and so did not require ethics approval.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by C Borner
Supplementary information
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Huang, A.S., Chin, H.S., Reljic, B. et al. Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins. Cell Death Differ 30, 632–646 (2023). https://doi.org/10.1038/s41418-022-01067-z
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/s41418-022-01067-z
This article is cited by
-
A new era in cancer therapy: targeting the Proteasome-Bcl-2 axis
Journal of Experimental & Clinical Cancer Research (2025)
-
March5-mediated Trim28 degradation preserves islet β-cell function in mice
Nature Communications (2025)
-
Inhibition of BAK-mediated apoptosis by the BH3-only protein BNIP5
Cell Death & Differentiation (2025)
-
Mitochondria and cell death
Nature Cell Biology (2024)
