1. Volume 36, Issue 4, Pages 696-703 (November 2009)
Bak Activation for Apoptosis Involves Oligomerization of Dimers via Their α6 Helices 
Grant Dewson, Tobias Kratina, Peter Czabotar, Catherine L. Day, Jerry M. Adams, Ruth M. Kluck 
Molecular Cell 
Volume 36, Issue 4, Pages (November 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Upon Apoptotic Signaling Bak α6 Helices Participate in Oligomerization
(A) Intermolecular C14:C14 and C166:C166 disulphide bonds can be induced in oligomerized Bak. Membrane fractions from bak−/− bax−/− MEFs expressing WT Bak (C14/C166) or the indicated cysteine variants were incubated with or without tBid, prior to treatment with the oxidant CuPhe (upper panel) or the crosslinker BMH (lower). Uncrosslinked Bak (M; monomer) and intramolecular (Mx) and intermolecular (D; dimer) crosslinked Bak were detected following SDS-PAGE (nonreducing for CuPhe samples) and immunoblotting for Bak.
(B) Position of single cysteine residues placed in the Bak α6 helix. Representation of the described cBak structure (Moldoveanu et al., 2006), with the α2 (red; BH3 domain), α3–4 (green; hydrophobic surface groove) and α6 (purple) helices indicated. Several residues that were mutated to cysteine (and C166) to test for oligomerization are indicated (sticks). Image was generated in PyMol.
(C) Cysteine substitutions in α2–6 do not disrupt proapoptotic function. bak−/− bax−/− MEFs expressing the indicated mutants were treated with UV (100 J/m2) and incubated for 24 hr. Percentage cell death relative to WT Bak is expressed as the mean ± SEM of three independent experiments.
(D) An α6:α6 dimer interface is evident only after tBid treatment. Membrane fractions from MEFs expressing WT Bak or the indicated Bak mutants were incubated with or without tBid, treated with CuPhe, and examined as in (A).
(E) Schematic of Bak conformation before and after apoptotic signaling. Bak in the nonactivated form is membrane anchored and can form intramolecular C14:C166 disulphide bonds (Mx). Upon apoptotic signaling, BH3 exposure allows the now-activated Bak to form a BH3:groove interface, possibly as symmetric dimers (Dewson et al., 2008). An α6:α6 interface (purple helices) then links symmetric dimers to form high-order oligomers.
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3. Figure 2
High-Order Oligomers of Activated Bak Contain Symmetric BH3:Groove Dimers Linked by α6:α6 Interactions
(A) Double and triple cysteine substitution in Bak does not disrupt proapoptotic function. bak−/− bax−/− MEFs expressing WT Bak or the indicated mutants were treated with etoposide and incubated for 24 hr. Percentage cell death is expressed as the mean ± SEM of at least three independent experiments.
(B) BH3:groove and α6:α6 interfaces are distinct and, when combined, allow detection of large oligomers. Membrane fractions from bak−/− bax−/− MEFs expressing WT Bak (C14/C166) or the indicated cysteine variants were incubated with or without tBid, prior to treatment with CuPhe. Samples were then run on 4%–20% SDS-PAGE under nonreducing conditions, or boiled in the presence of 5% v/v 2-mercaptoethanol for reducing conditions, and assessed as in Figure 1A. Note that BH3:groove dimers (D1) migrate differently to H164C:H164C dimers (D2).
(C) Schematic of oligomerized Bak and the complexes detected following disulphide bonding of double-, single-, and triple-cysteine variants. A Bak hexamer containing symmetric BH3:groove dimers linked by α6:α6 interactions is represented as in Figure 1E. Following oxidation with CuPhe, nonreducing SDS-PAGE reveals disulphide-bonded dimers (D1 and D2) or large oligomers, depending on the positions of introduced cysteine residues (red for BH3, green for groove, purple for α6).
(D) Some Bak oligomers exceed 18X complexes. Samples from (B) were run on nonreducing 3%–8% SDS-PAGE and assessed as in Figure 1A.
(E) Formation of the α6:α6 interface is dependent on the BH3:groove interface. Membrane fractions from bak−/− bax−/− MEFs expressing the indicated cysteine variants of Bak were incubated with tBid in the presence or absence of the BH3-specific (4B5) or 7D10 anti-Bak rat monoclonal antibodies. Samples were then treated with CuPhe and assessed as in Figure 1A.
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4. Figure 3
N- and C-Terminal Truncation of Bak Is Not Sufficient to Provoke an α6:α6 Interface
(A) The 8F8 epitope contains residues within hBak 8–17. Lysates of MEFs expressing WT Bak or N-terminal truncated Bak (ΔN7 and ΔN17) were run on SDS-PAGE and triplicate blots probed with the 8F8, Ab-1, and 7D10 anti-Bak antibodies.
(B) 8F8 and Ab-1 epitopes become exposed with Bak activation. Membrane fractions from MEFs expressing WT Bak were incubated with or without tBid and then immunoprecipitated with the indicated anti-Bak antibodies in either digitonin or Triton X-100. Immunoprecipitates were blotted for Bak.
(C) Schematic of human Bak truncation mutants analyzed in (D–F). α helices 1–9 are indicated, as are the Bak regions recognized by the 8F8 and Ab-1 antibodies, and the BH3 and transmembrane (TM) domains.
(D) Truncation of the C terminus but not N segment abrogates Bak proapoptotic function. bak−/− bax−/− MEFs expressing WT Bak or the indicated truncation mutants were assessed for Bak protein by immunoblotting (lower panel). Cells were also treated with UV (100 J/m2) and incubated for 24 hr, with percentage cell death expressed as the mean ± SEM of three independent experiments (upper panel).
(E) Truncation of the N segment alone does not allow an α6:α6 interface. Membrane fractions from MEFs expressing WT Bak or the indicated Bak mutants were incubated with or without tBid, treated with CuPhe, and assessed as in Figure 1A. Each variant retained proapoptotic function (Figures 1C and 3D; data not shown).
(F) Truncation of the C terminus abrogates Bak membrane insertion and conformation change. MEFs expressing the indicated Bak variants were separated into membrane (Mem) and cytosolic (Cyt) fractions, then incubated with or without tBid, treated with CuPhe, and assessed as in Figure 1A.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Mutations to the Putative Zinc-Binding Site Do Not Affect Bak Function
(A) Bak zinc-binding site single-point mutants are stably expressed and retain proapoptotic function. bak−/− bax−/− MEFs were retrovirally infected with WT Bak or the indicated mutants, and cell lysates were immunoblotted for Bak and reprobed for HSP70 as a loading control. Cells were also treated with graded doses (0, 0.1, 1, and 10 μM) of etoposide for 24 hr, with percentage cell death expressed as the mean of two independent experiments.
(B) WT Bak and zinc-binding site single-point mutants change conformation and oligomerize after tBid treatment. Membrane fractions expressing the indicated Bak mutants were incubated with or without tBid, treated with CuPhe, and assessed as in Figure 1A.
(C) The α6:α6 interface in activated Bak does not require the putative zinc-binding site in α6. Membrane fractions expressing the indicated Bak mutants were incubated with or without tBid, treated with CuPhe, and assessed as in Figure 1A.
(D) Zinc inhibits Bak-mediated cytochrome c release, and Bak conformation change and oligomerization, even if the putative zinc-binding site is mutated. Membrane fractions expressing the indicated Bak mutants were treated with tBid plus the indicated concentration of ZnCl2 for 30 min at 30°C. Supernatant (S) and pellet (P, membrane) fractions were separated and immunoblotted for cytochrome c (left). In addition, membrane fractions were treated with CuPhe, and assessed as in Figure 1A (right).
Molecular Cell  , DOI: ( /j.molcel )
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