Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function Pedro Eitz Ferrer1¤a, Paul Frederick1¤b, Jacqueline M. Gulbis1,2, Grant Dewson1,2., Ruth M. Kluck1,2.* 1 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 2 Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia Abstract Background: One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear. Methodology/Principal Findings: To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C- terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1. Conclusions/Significance: Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax. Citation: Ferrer PE, Frederick P, Gulbis JM, Dewson G, Kluck RM (2012) Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function. PLoS ONE 7(3): e31510. doi:10.1371/journal.pone.0031510 Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America Received May 26, 2011; Accepted January 11, 2012; Published March 19, 2012 Copyright: 2012 Ferrer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grant #10-230 from the AICR http://www.aicr.org.uk and grants #637335 and #637337 from the National Health and Medical Research Council www.nhmrc.gov.au, as well as Future Fellowships (GD and RK) from the Australian Research Council http://www.arc.gov.au, and was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. ¤a Current address: Institute for Microbiology, Immunology and Hygiene, Technische Universita¨t M u¨nchen, Munich, Germany ¤b Current address: Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia * E-mail: kluck@wehi.edu.au Introduction The intrinsic or mitochondrial pathway of apoptosis is regulated by the Bcl-2 protein family, with two members, Bak and Bax, required to permeabilize the mitochondrial outer membrane (OM) [1,2]. During OM permeabilization, Bak and Bax undergo significant conforma- tion change involving exposure of N-terminal epitopes and homo- oligomerization [3,4,5,6,7] to form an as yet undefined pore. Bak conformation change also involves transient exposure of the BH3 domain that then binds to the hydrophobic groove of another activated Bak molecule to form symmetric dimers [8,9,10], with the same process also evident for Bax [11,12,13,14]. Symmetric dimers of Bak and of Bax can then be linked by an a6:a6 interface into higher order oligomers that likely constitute the apoptotic pore [8,12]. Bak and Bax are regulated by other Bcl-2 family members. They are activated by direct binding of BH3-only proteins (e.g. Bim and tBid), and sequestered by binding to prosurvival proteins (e.g. Mcl-1 and Bcl-2) [15,16,17,18,19,20]. Specific binding results in Bak being guarded mainly by Bcl-xL, Mcl-1 and A1, while Bax is countered mainly by Bcl-2, Bcl-xL, Bcl-w and A1 [20,21,22,23]. This specific binding can result in either Bak or Bax preferentially driving apoptosis [24,25]. For example, Bak-driven apoptosis can be initiated by loss of the relatively labile Mcl-1 and Bcl-xL following UV, actinomycin D or cycloheximide [20,26]. Bak and Bax contain a C-terminal hydrophobic region that inserts as a transmembrane (TM) domain into the mitochondrial OM. The C-termini of Bak and Bax can target GFP to mitochondria [27,28], and their truncation in the native proteins PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e31510