the dynamic interplay between pro- and antiapoptotic members of the bcl-2 family dictates life and death decisions .
activation of the proapoptotic bcl-2 proteins , bak and bax ( the effectors ) , represents the pivotal step toward apoptosis by mediating mitochondrial outer membrane permeabilization ( momp ) ( chipuk and green , 2008 ; youle and strasser , 2008 ) . despite their essential role in apoptosis , how effectors are activated by other bcl-2 family members
according to the indirect model , antiapoptotic bcl-2 proteins sequester effectors to block their proapoptotic function .
bh3-only proteins can displace bak or bax from their antiapoptotic counterparts , thereby facilitating their oligomerization and subsequent momp ( willis et  al . , 2005 ) .
a competing direct activation model proposes that effector function is triggered by activator bh3-only proteins , such as bim and tbid ( kim et  al . , 2009 ;
although the underlying mechanism of activation remains unclear , effectors undergo major structural reorganizations in order to convert the monomeric , globular , closely packed protein into a highly ordered , multimeric mitochondrial permeabilizing pore .
the association of bak with antiapoptotic bcl-2 family members , as predicted by the indirect model , occurs through the canonical bh3:groove interface ( sattler et  al . , 1997 ;
because the bh3 domain is occluded in inactive bak , exposure of the bh3 domain , which coincidentally results in the opening of the hydrophobic binding groove ( bh1bh3 domains ) , has to precede bcl - xl association ( moldoveanu et  al . , 2006 ) .
this conformational change may be induced by direct association with activators either through the canonical bh3:groove interface or the bh3 trigger site ( a hydrophobic cleft opposite the canonical groove formed by 1 and 6 helices ; czabotar et  al . , 2013 ;
the resulting conformation can then engage the bh3 domain of a neighboring effector protein to form a reciprocal , symmetric , bh3:groove dimer ( dewson et  al . , 2008 ) .
the assembly into high - molecular - weight complexes occurs through a second 6:6 interface ( dewson et  al . ,
in addition , based on a structural similarity to -pore - forming toxins ( -pfts ) , execution of effector function may require membrane insertion of the central 5/6 hairpin ( pore domain ) to form the mitochondria - permeabilizing pore ( annis et  al .
1996 ) . to further delineate and characterize the steps required for the transition of native bak into a membrane - permeabilizing pore , we combined biochemical and structural approaches to reveal a progressive sequence of ( 1 ) exposure of the bh3 domain , ( 2 ) n - terminal change , ( 3 ) oligomerization , and ( 4 ) insertion of the pore domain . by establishing a  bcl - xl addiction
release of this checkpoint by abt-737 facilitates direct association with bim promoting membrane insertion and subsequent cell death .
although the activation of bak may occur either directly or indirectly , a key feature of both models is the neutralization of antiapoptotic bcl-2 proteins by bh3-only proteins . to mimic this , we used abt-737 , which binds with high affinity to the hydrophobic groove of bcl - xl , bcl-2 , and bcl - w and induces apoptosis in a bak and/or bax - dependent manner ( oltersdorf et  al . , 2005 ; van delft et  al . , 2006 ; vogler et  al . ,
jurkat cells exposed to abt-737 exhibited classical apoptotic characteristics , including reduction in m , increased phosphatidylserine ( ps ) externalization , cytochrome c release , and processing of caspase-3 and poly ( adp - ribose ) polymerase ( parp ) ( figures 1a and 1b ) .
as these cells lack bax , abt-737-induced apoptosis would be predicted to be solely dependent on bak ( shawgo et  al . , 2008 ) .
in agreement with this , suppression of bak expression resulted in a marked decrease in ps externalization following abt-737 treatment ( figure  1c ) . according to the indirect activation model , bak
is held in check by antiapoptotic proteins ( ruffolo and shore , 2003 ; willis et  al . , 2005 ) .
immunoprecipitations revealed that bak was associated with bcl - xl , but not bcl-2 or mcl-1 in jurkat cells ( figure  1d ; data not shown ) .
abt-737 antagonized bcl - xl and efficiently displaced bak compatible with features of the indirect model .
closer analysis of the supernatant fractions indicated that , although the target protein was completely depleted , a reciprocal reduction in its coprecipitated binding partner was not observed ( figure  1d , box ) .
thus , only a discrete portion of bak appeared to be complexed with bcl - xl .
the direct activator bim was also released from bcl-2 and bcl - xl by abt-737 , and although its displacement was less efficient compared to bak , released bim could potentially act to  directly  activate inactive bak ( figures 1e , 1f , and s1 ) . if the release of bak from bcl - xl is functionally important for abt-737-induced apoptosis , then introduction of bcl - xl : bak complexes into resistant cells should sensitize them to abt-737 .
to exclude interference from endogenous bak or bax , bcl - xl : bak complexes were introduced into bax and bak double - knockout ( dko ) mouse embryonic fibroblasts ( mefs ) . as coexpressing bak with bcl - xl resulted in cell death , we first expressed bcl - xl followed by tetracycline - induced expression of bak ( figure  2a ) . upon induction ,
sink  of bcl - xl resulting in cells with bak precomplexed to bcl - xl exclusively .
reconstitution of bcl - xl : bak complexes sensitized dko mefs to abt-737 as assessed by ps externalization and processing of caspase-9 ( figure  2b ) . as in this model system
, all induced bak was sequestered , effectively  addicting  the cells to bcl - xl ( figure  2c , box ) , and abt-737-induced cell death was a result of bak release from bcl - xl .
reconstitution of cells with mcl-1:bak did not confer sensitivity to abt-737 validating our model system ( figures s2a and s2b ) .
introduction of bcl - xl : bim or bcl-2:bim complexes into human embryonic kidney 293 t ( hek293 t ) cells did not alter sensitivity to abt-737 ( figures 2d and 2e ) .
thus , bim released from bcl-2 or bcl - xl did not appear to exert a direct activator function on endogenous bak or bax , further suggesting that the reconstituted bcl - xl : bak complexes are the preferential target of abt-737 .
bak associates with bcl - xl through the canonical bh3:groove association ( sattler et  al . , 1997 ) . in agreement ,
introduction of bak bh3 mutants l78a or d83a into the reconstitution system revealed that , whereas both coimmunoprecipitated with bcl - xl , they were , in contrast to wild - type ( wt ) bak , also recovered from the supernatant ( figure  2f ) .
this implied both mutants displayed a reduced affinity for bcl - xl and confirmed the requirement of the bh3 domain for complex formation .
as this domain is occluded in inactive bak , bcl - xl sequesters a preactivated , bh3-domain - exposed conformation .
in addition to bh3 domain exposure , conformational changes within the n - terminal region are also characteristic of bak activation and are commonly determined by exposure of the ab-1 epitope and increased trypsin sensitivity ( griffiths et  al .
limited trypsin proteolysis on mitochondria isolated from abt-737-treated jurkat cells revealed a bak cleavage fragment ( p19 ) , which was only detectable with the gly82 antibody ( epitope in the bh3 domain ) .
in contrast , antibodies directed against the n - terminal region ( nt and ab-1 ) revealed a reduction in full - length bak without detection of the p19 fragment , linking trypsin sensitivity to n - terminal change ( figures 3a , 3b , and s3 ) .
together with formation of the p19 fragment , ab-1-associated fluorescence was also detectable and occurred in the entire cell population , but not when cells were treated with an inactive enantiomer of abt-737 ( figure  3c ) .
thus , n - terminal change required the neutralization of antiapoptotic proteins by abt-737 and , by inference , this must occur downstream of bak displacement from bcl - xl . to further explore the relationship between displacement and n - terminal change ,
we established a double immunoprecipitation procedure : the first was directed against ab-1-exposed bak , followed by a second , on the resulting supernatant , against bcl - xl ( figure  3d ) .
when jurkat cells were lysed in chaps ( to retain bak in its native conformation ) , ab-1 failed to precipitate bak and the bcl - xl : bak complex was recovered from the resulting supernatant ( figure  3e , box ) .
thus , bak complexed with bcl - xl had not yet exposed the ab-1 epitope .
abt-737 treatment resulted in the recovery of bak by ab-1 , but bcl - xl failed to coprecipitate suggesting that bak exposes this epitope downstream of its release from bcl - xl ( figure  3e ) .
this was confirmed by the second immunoprecipitation , which recovered bcl - xl without associated bak . taken together , these data demonstrated that the bak n - terminal change occurred downstream of its displacement from bcl - xl .
as the bak bh3 domain is required for association with bcl - xl , this further inferred that eversion of the bh3 domain occurred independent of and upstream of the n - terminal change .
in addition , when the n terminus of bak was artificially altered by triton x-100 , the bcl - xl : bak complex was recovered in both immunoprecipitations ( figure  3e ; dewson et  al . , 2008 ) .
thus , under these conditions , bcl - xl - sequestered bak  existed as a heterogeneous population of bh3-domain - exposed / ab-1-exposed as well as bh3-domain - exposed / ab-1-occluded conformations .
identical mixed conformations were also obtained by double immunoprecipitation with reconstituted cells solubilized in chaps and abt-737 treatment , which resulted in conversion to an exclusively n - terminal changed population ( figure  3f ) .
thus , the n terminus can be altered either artificially with detergents or by  overexpression  without affecting the bh3:groove association highlighting the independence of both events .
the oligomerization of bak into dimers and high - molecular - weight complexes is a prerequisite for momp and can be assessed using the redox catalyst copper(ii)(1,10-phenanthroline)3 ( cuphe ) ( dewson et  al . , 2008 ; falke et  al . ,
1988 ) . in untreated jurkat cells , cuphe induced the formation of an intramolecular disulfide bond between n - terminal c14 and 6-localized c166 resulting in a faster migrating band on sds - page ( mx ) ( figure  4a , left panel ) .
abt-737 treatment shifted the mx form into disulfide - linked dimers ( d ) and higher molecular weight complexes ( d ) .
reduction of disulfide bonds resulted in the detection of solely monomeric bak ( figure  4a , right panel ) .
oligomerization involves two distinct interfaces , the bh3 domain / groove and the 6 helix ( dewson et  al . ,
as the 6 helix is occluded by 1 helix in inactive bak , the n - terminal change has to precede oligomerization . to analyze the chronology of the n - terminal change and oligomerization , bak was restrained in its mx conformation prior to activation .
after incubation at 42c to activate bak ( pagliari et  al . , 2005 )
, oligomer formation as well as cytochrome c release was reduced compared to  unrestrained  bak ( figures 4b and 4c ) .
these results indicated that the n - terminal change must precede oligomerization possibly to enable exposure of the oligomerization interface , the 6 helix , to the protein surface .
interference with the bh3:groove interface by utilizing a bak l78a or an n - terminal - truncated bak n83 , which lacks a major part of the bh3 domain , did not impair oligomer formation ( figures 4d and s4b ) . despite undergoing oligomerization
, l78a bak failed to induce apoptosis as assessed by ps externalization and cytochrome c release ( figures 4e , 4f , and s4a ) .
as l78a bak also underwent  n - terminal change , a prerequisite for oligomerization ( figure  4f ) .
therefore , the ability to oligomerize did not appear to determine the proapoptotic activity of bak suggesting the requirement of additional steps to convert bak into a membrane - permeabilizing pore .
membrane insertion of the bax pore domain is associated with  the formation of a momp - inducing pore ( annis et  al . , 2005 ;
we hypothesized that , following oligomerization , bak inserts its pore domain  into the membrane and the l78a mutant may be blocked at this stage accounting for its inactivity .
the membrane topology of the  5/6 hairpin in wt versus l78a  bak was analyzed by combining cysteine mutagenesis with 4-acetamido-4-((iodoacetyl ) amino ) stilbene-2,2-disulfonic acid ( iasd ) labeling ( annis et  al . , 2005 ; krishnasastry et  al . ,
iasd only labels sulfydryl groups exposed to a hydrophilic environment resulting in the labeling of cysteine residues in the cytosol , the cytoplasmic boundary of a pore ,  or those facing the lumen of an aqueous pore .
cysteines  embedded within the lipid bilayer or buried within a protein  interface will be protected from iasd labeling ( figure  5a ) .
cysteines were introduced in the pore domain of wt and l78a bak using cysteine - deficient bak ( c14a / c166a ) as a template .
insertions did not appreciably affect proapoptotic activity when overexpressed in dko mefs ( figure  s5 ) .
as bak is constitutively membrane - anchored , a cysteine insertion at residue l195 within the transmembrane region ( 9 helix ) served as a control .
following overexpression in dko mefs , both wt and l78a l195c exhibited an unaltered mobility on sds - page compared to the unlabeled control ( figure  5b ) .
this protection from iasd labeling represented membrane insertion , as solubilization in chaps resulted in an iasd - mediated increase in molecular weight .
in contrast , the n - terminal endogenous cysteine c14 in both wt and l78a was labeled by iasd indicating the constitutive cytosolic localization ( figure  5b ) .
if the killing conformations of bak and bax are analogous , then the 5/6 hairpin should be embedded in the membrane and , as a result , cysteines within these helices would be expected to be protected from iasd labeling ( annis et  al . , 2005 ) .
conversely , if membrane insertion of the l78a mutant is impaired , then the corresponding cysteines would remain cytosolic and thus modified by iasd .
in contrast a cysteine insertion at n124 ( first n - terminal residue of the 5 helix ) as well as the endogenous cysteine c166 ( on the c - terminal end of the 6 helix ) were modified by iasd in both wt and l78a bak ( figure  5b ) .
however , as both cysteines localize to the extremities of the 5 and 6 helices in wt bak , they could represent the cytoplasmic boundary of a membrane - inserted pore domain ( figure  5a ) .
cysteine residues with a more centralized localization within the 5/6 helices should therefore be protected in the  killing conformation  of wt bak . despite this , residues r137c
( 5 helix ) as well as wt v154c and r156c ( both 6 helix ) were also labeled by iasd ( figure  5b ) . however , the labeling pattern of the corresponding l78a mutants differed by the presence of an iasd - protected species .
solubilization with chaps resulted in a complete shift to a labeled band , thus excluding the occlusion of these cysteines in a protein - protein interface and demonstrating there was sufficient iasd for labeling .
these results indicated that the pore domain of l78a adopts a different membrane topology to wt bak possibly explaining opposing apoptotic activities of wt and l78a bak .
the different membrane topologies of wt compared to l78a bak imply that adoption of the killing conformation was dependent on the bh3 domain . as the bh3 domain is part of the groove , the nonkilling , iasd - protected conformation of l78a could result from impaired engagement of a  ligand  bh3 domain which can be provided by a direct activator . in support of this hypothesis , we found that coexpression of the direct activator bim with wt , but not l78a bak , promoted cell death ( figure  6a ) .
this synergism correlated with the coimmunoprecipitation of bim with wt bak , but not with the l78a mutant ( figure  6b ) . in this coexpression approach , wt bak is already oligomerized when transfected alone indicating a role of bim downstream of oligomerization possibly by promoting membrane insertion ( figure  6c ) . in agreement , bim altered the membrane topology of wt r137c or v154c by inducing a shift from the unlabeled to the iasd - labeled species ( figure  6d , box ) .
however , both l78a and wt bak gave a double band which was associated with cell death only with wt , but not l78a bak .
a similar pattern was also observed when wt bak was overexpressed at an intermediate killing concentration ( figure  s6a ) .
thus , the double band obtained with wt and l78a bak may represent different membrane topologies . to further investigate the effects of bim on bak , bim expression was knocked down in the reconstitution system .
reduction of bim expression resulted in a decrease of abt-737-induced cell death ( figure  6e ) .
we hypothesized that this effect should correlate with retention of wt r137c and v154c bak in an iasd - protected nonkilling conformation ( figures 6d and s6a ) . agreeing with this ,
wt r137c was predominantly iasd - labeled and suppression of bim resulted in reversion of the  labeled to an unlabeled species which was independent of release from bcl - xl by abt-737 ( figure  6f ) . when reconstituted , bcl - xl sequestered oligomeric bak and thus  this does not contradict the proposed order of oligomerization followed by membrane insertion ( figures s1 , 4a , and s6b ) .
wt v154c gave a mixture of labeled and unlabeled species , and suppression of bim reduced the iasd - labeled species , although this effect was only evident following abt-737 treatment ( figure  6f , box ) .
taken together , these  data revealed that the proapoptotic activity of wt bak  correlated with the conversion of an iasd - protected to a  labeled conformation , which was promoted by interaction with bim .
bak and bax are essential effectors of momp , the point of no  return in apoptotic cell death , and as a consequence , their activation must be highly regulated . in this study , we demonstrate that activation of bak involves several sequential but independent conformational changes , which provide different regulatory checkpoints mediated by members of the bcl-2 family .
the antiapoptotic arm of this family functions to sequester a bh3-domain - exposed conformation of bak preventing its further activation .
bh3 mimetics , such as abt-737 , can release this checkpoint facilitating n - terminal change and oligomerization .
the ability to oligomerize , however , did not correlate with cell death , and formation of a momp - inducing pore required association with the direct activator bim . in agreement with the indirect activation model
, we validated a role for bcl - xl and mcl-1 , but not bcl-2 , in inhibiting bak activation ( ruffolo and shore , 2003 ; wei et  al .
, 2000 ; willis et  al . , 2005 ; figures 1 , 2 , and s2 ) .
bak associates with bcl - xl through the canonical bh3:groove interface , which is stabilized by hydrophobic and electrostatic interactions .
interference with either ( l78a or d83a bak bh3 mutants ) reduced binding to bcl - xl ( figures 1d , 2c , and 2f ; sattler et  al . , 1997 ) .
by competing for the groove of bcl - xl , abt-737 displaced bak from bcl - xl further establishing the importance of the bh3:groove association ( figures 1d and 2c ; oltersdorf et  al . ,
2005 ) . in inactive bak , key stabilizing hydrophobic residues within the bh3 domain face
the protein core and association with bcl - xl requires rotation of the bh3 domain - containing 3 helix to evert  these residues to the protein surface .
this change involves the opening of the otherwise occluded hydrophobic groove ( bh1bh3 domains ) .
thus , bcl - xl does not sequester inactive bak but a preactivated , bh3-domain - exposed / open - groove conformation .
in contrast to bak , in bax the 9 helix ( transmembrane anchor ) occludes the groove / bh3 domain .
its disengagement is required to expose the bh3 domain / groove , which may be allosterically induced by association of activators with the bh3 trigger side to induce an n - terminal change ( cartron et  al . , 2004 ; gavathiotis et  al . ,
thus , for bax , n - terminal change precedes exposure of the bh3 domain / groove which is accompanied by translocation to the mitochondria . as bak
constitutively localizes to the mitochondria and the 1/6 binding site is not as pronounced , it has been proposed that bak can bypass this activation step resulting in constitutive exposure of the n terminus ( kim et  al . , 2009 ) .
exposure of a cryptic antibody epitope ( ab-1 ) , increased trypsin sensitivity , as well as the loss of the mx form are associated with the n - terminal change and exclusively occurred following abt-737 treatment ( griffiths et  al . , 1999 ;
wei et  al . , 2000 ; figures 3b , 3c , and 4d ) . by mapping the ab-1 epitope to y38 to the middle of the 1 helix
, we attribute the increased accessibility to this epitope to a major reorganization of the n - terminal region ( 1 and 2 helices ) rather than the simple exposure ( figure  s3 ) . however , despite the close proximity of the n - terminal region with the bh3 domain , the n - terminal change occurred independent and downstream of bh3 domain exposure as bcl - xl - sequestered bak existed exclusively in an n - terminal - occluded conformation ( figures 3e and 3f ) .
furthermore , the n - terminal change is a prerequisite for oligomerization , as the formation of oligomers could be blocked by inhibiting the n - terminal change ( figures 3c , 4a , and 4b ) .
this supports the proposed model for oligomerization as the n terminus occludes the 6 helix which represents the interface for bh3:groove - linked dimers to associate into oligomers ( dewson et  al . , 2008 , 2009 ) .
however , the apparent , indispensable role of the bh3 domain in oligomer formation is unclear as both bakn83 as well as bak l78a both form oligomers ( figures 4d and s4b ) .
although bak is oligomeric in the reconstitution system , bh3 domains appear to be available both to facilitate a bh3:groove association with bcl - xl and to allow interaction with bim ( figures 6b and s6b ) . despite this
, the bh3 domain clearly contributes to bak proapoptotic function , as both bakn83 as well as l78a lost their killing ability ( figures 4e and s4a ) .
thus , in analogy to bax , a bh3-domain - dependent step must occur downstream of oligomerization ( kushnareva et  al . , 2012 ) .
structural similarities between -pfts prompted the hypothesis that bak and bax insert into the membrane to mediate momp ( muchmore et  al .
the membrane - inserting domains of -pfts are composed of a hydrophobic , central , helical hairpin which , in the native conformation , is surrounded by amphipathic helices . during activation ,
a series of conformational changes opens up these outer layers to expose the central hairpin , which then spontaneously penetrates the membrane ( parker et  al . ,
1990 ) . by analogy , during bak activation , the outer 1 helix repositions away from the protein core exposing the 6 helix , which together with the 5 helix resembles the central hairpin of the pfts . however , the pore domain of bak is comprised of largely amphipathic helices , which are not expected to spontaneously membrane insert .
the occlusion of polar surfaces within tertiary contact sides may energetically favor membrane insertion ( walker et  al .
by analogy , bak assembled into high - molecular - weight complexes prior to membrane insertion ( membrane anchored conformation ; figure  s7i ) and consequently could be labeled by iasd ( figures 5b and 6d ; labeled band with l78a r137c and v154c ) .
this effectively concentrated these subunits on the membrane surface potentially facilitating the coordinated membrane insertion ( figure  s7ii ) .
evidence for a membrane - inserted conformation was suggested by the protection of r137c and v154c from iasd when using sublethal concentrations of wt bak ( figure  6d ) or in l78a bak ( figures 5b and 6d ) . however , this labeling pattern was not associated with killing and consequently could not represent the final pore but rather a prepore conformation ( figures s6 and s7ii ) .
in contrast , when wt bak r137c and v154c were expressed at killing concentrations , they were accessible to iasd ( figure  5b ) . considering that in active bak the 5 and 6 helices have to adopt a transmembrane / membrane - inserted conformation ( oh et  al . , 2010 ) , accessibility to iasd
could be explained by the localization of these residues to the lumen of an aqueous pore . in support of this ,
amphipathic helices , like the pore domain of bak , were shown to orientate their polar patches toward the lumen of the pore to expose nonpolar surface to the hydrophobic core of the bilayer ( gonzlez - maas et  al .
bax is also described to form an ion - conductive , aqueous pore implying that polar residues within the pore domain must orientate to the lumen of the pore  ( antonsson et  al . , 1997 ; schlesinger et  al . , 1997 ) .
thus , several lines of evidence point toward a membrane - inserted pore domain lining the lumen of an aqueous momp pore .
this provides a possible explanation for the observed shift from unlabeled to labeled conformations which correlated with cell death .
the pore domain seemed to reorientate within the membrane from a state where the pore domain was  partially  inserted into the membrane ( iasd - protected ) to the mature momp - inducing pore ( iasd - labeled ) . as pore formation appeared to be a highly dynamic process ( figure  s6a ) , a continuous flux from membrane - anchored to pore conformation could occur , possibly explaining the detection of double bands under certain conditions .
multidomain bcl-2 family members , such as bak , are capable of acting both as ligand or receptor .
inserting its bh3 domain into the hydrophobic binding groove of antiapoptotic bcl-2 proteins affords bak ligand characteristics , whereas the structurally juxtaposed bh1bh3 domains form a hydrophobic groove , which can act as a receptor for the bh3 domain of direct activator proteins ( czabotar et  al . , 2013 ; kim et  al . , 2009 ; leshchiner et  al . ,
activator engagement promotes  the exposure of the effector bh3 domain and opening of the groove , thereby creating the interfaces required for oligomerization . in our model , we could not find any direct activator role of bim on endogenous bak or bax following its release from bcl - xl ( figure  2d ) .
however , the importance of direct activators in the initial activation of both bak and bax has been demonstrated recently using  bh3-stabilized  helices of bcl-2 proteins  or bh3 peptides on recombinant full - length or truncated effectors ( czabotar et  al .
2013 ) . in agreement , bim synergized with bak proapoptotic function as coexpression of both proteins enhanced and vice versa and decrease of bim levels reduced cell death ( figures 6a and 6e ) .
this effect was only evident with wt , but not l78a bak , implying the requirement of an intact receptor surface to accommodate the bh3 domain of the activators .
the direct activator role of bim is attributed to the induction of bak oligomerization ( cheng et  al . , 2001 ; kim et  al . , 2009
however , in both model systems used , coexpression or reconstitution , bak was oligomerized prior to bim association ( figures 6c and s6b ) .
although we can not exclude a possible involvement of bim in the initial activation of bak , our results suggest bim can also act downstream of oligomerization in an unappreciated role to convert the membrane topology of oligomerized , partially membrane - inserted prepore bak to the killing pore conformation .
this hypothesis is supported by our finding that coexpression of bim shifted wt r137c and v154c from an iasd - protected to a labeled species , whereas knockdown of bim reversed this ( figures 6d and 6f ) .
the n - terminal part of the 5 helix ( amino acids 124136 ) comprises the bh1 domain and thus participates in groove formation .
complete membrane insertion of this helix would effectively destroy the hydrophobic groove causing a loss of receptor characteristic .
this would suggest that in prepore bak , protection from iasd - labeling may represent the partial insertion of the 5 helix to maintain the structure and function of the groove and thus facilitate engagement with the bim bh3 domain ( figures 6d and 6f ) .
bim could then promote conformational changes within the groove leading to complete insertion of the pore domain . in summary
, our results reveal that bak activation is a complex process that includes a series of independent steps ( figure  7 ) .
although bak adopts an oligomeric conformation after its release from bcl - xl , this conformation does not induce momp .
we have identified a requirement for the direct activator bim to convert oligomerized prepore bak into a membrane - permeabilizing pore .
therefore , bak activation does not exclusively occur through the direct or indirect activation models but rather combines aspects of both .
these additional steps provide further unappreciated checkpoints to control activation and prevent unwanted / accidental cell death and also open up possible opportunities for tumors to deregulate apoptosis .
detailed description of cell culture , immunoprecipitation , limited proteolysis , ab-1 staining , transfection methods , and cell death analysis are provided in the extended experimental procedures .
human bak was cloned into pcdna4to with an n - terminal strep - tag ii .
strep - flag double - tagged bcl - xl and bimel were cloned into pcdna6 .
a. strasser and d. huang , the walter and eliza hall institute of medical research ) using jetprime ( polyplus ) or hek293 t cells using jet pei ( polyplus ) according to manufacturer s protocol .
where indicated , site - directed mutagenesis was performed using quikchange multi ( agilent technologies ) , and all constructs were sequence - verified prior to use .
cells were transiently transfected with bak , tetr , and bcl - xl constructs at ratios 1:10 ( bak : tetr ) and 1:1 ( bak : bcl - xl ) .
bak was induced with tetracycline ( 4  g / ml ; calbiochem ) at 24  hr posttransfection for 4  hr prior to treatment with abt-737
. formation of bak high - molecular - weight complexes was analyzed using the redox catalyst cuphe .
mitochondria - enriched fractions from jurkat cells or digitonin - generated heavy membrane fractions from mefs were solubilized in x - link buffer ( 100  mm sucrose , 20  mm hepes - koh [ ph 7.4 ] , 2.5  mm mgcl2 , and 50  mm kcl ) and incubated with cuphe ( 20  mm 1,10 phenanthroline with 300  mm cuso4 ) for 30  min on ice .
the reaction was then quenched by incubation with 100  mm edta for 15  min and mitochondria - enriched fractions recovered .
bak cysteine insertion mutants were transiently transfected into dko mefs or  hek293 t cells .
heavy membrane fractions were obtained by mild digitonin lysis and washed with 10  mm dithiothreitol ( dtt ) to ensure reduction of sulfydryl groups .
pellets were resuspended in x - link buffer and incubated with 10  m iasd for 30  min at room temperature .
reactions were quenched by addition of 100  mm dtt and mitochondria - containing fractions recovered .
resulting pellets were solubilized in 2% chaps and iasd modification analyzed by sds - page followed by western blotting on large - format sds - page gels . as controls ,
samples were left unlabeled or lysed in 2% chaps prior to iasd labeling . extended experimental procedurescell culturecell lines were obtained from the european collection of animal cell cultures ( ecacc ; porton down , uk ) except where indicated .
jurkat cells ( clone e6.1 ) were maintained in roswell park memorial institute ( rpmi ) medium containing 10% fetal calf serum ( fcs ) and 5% glutamax ( life technologies inc .
paisley uk ) , human embryonic kidney fibroblasts ( hek293 t ) and mefs deficient in bax and bak were cultured in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% ( fcs ) and 5% glutamax.cell death induction and apoptosis quantificationthe intrinsic apoptotic pathway was induced by treatment with abt-737 ( abbott laboratories , illinois , usa ) with the concentration used dependent on the cell type ( 3 - 30  m ) .
apoptosis was measured using annexin v and propidium iodide ( pi ) staining . for non - adherent cells , annexin buffer ( 10  mm hepes / naoh ( ph 7.4 ) , 150  mm nacl
, 5  mm kcl , 1  mm mgcl2 , 1.8  mm cacl2 ) supplemented with annexin v - fitc was added to the cells .
after incubation for 10  min , propidium iodide was added and cells analyzed on a facscan flow cytometer using cellquest software ( becton dickinson , oxford uk ) . for adherent lines , both detached and trypsinized cells were resuspended in fresh medium and left to recover prior to staining .
tmre ( 100  nm ) was added to medium and cells incubated at 37c for 10  min in the dark prior to analysis by flow cytometry .
both tmre and annexin v measurements were made on the whole cell population.analysis of bak n - terminal conformation 
cells were fixed with 2% paraformaldehyde at room temperature for 10  min , washed with pbs and resuspended in permeabilization buffer ( 0.1% saponin and 0.5% bsa in pbs ) containing bak ab-1 antibody ( calbiochem , nottingham , uk ) ( 1:100 dilution ) .
after incubation for 1  hr at 4c , cells were washed and incubated with goat anti - mouse igg - alexa fluor 488 ( invitrogen ) diluted 1:100 into permeabilization buffer .
after washing in pbs , cells were analyzed by flow cytometry.limited trypsin proteolysisfor limited trypsin proteolysis , mitochondria enriched fractions were resuspended in x - link buffer ( 100  mm sucrose , 20  mm hepes - koh ph 7.4 , 2.5  mm mgcl2 , 50  mm kcl ) and 125  g trypsin/50  g protein was added . following incubation for 20  min on ice trypsin
was inhibited by addition of 4  mm pefabloc sc ( roche diagnostics , basel , switzerland ) .
mitochondria enriched fractions were recovered and analyzed by sds-page.ab-1 staining and confocal microscopyfor immunofluorescent staining , cells were grown on coverslips and transfected as described .
after 24  hr , cells were fixed with 4% ( v / v ) paraformaldehyde and permeabilized with 0.5% saponin in pbs .
cells were then incubated with anti - cytochrome c and ab-1 followed by the appropriate fluorophore - conjugated secondary antibodies ( alexa 547 for cytochrome c and alexa 488 for ab-1 ) and hoechst 33342 .
coverslips were then mounted on glass slides and subjected to confocal microscopy on a zeiss lsm510.subcellular fractionationheavy membrane ( hm ) fractions were generated by resuspending in mitochondrial isolation buffer ( 250  mm sucrose , 20  mm hepes , ph 7.4 , 5  mm mgcl2 and 10  mm kcl ) containing 0.01% digitonin .
cells were left on ice followed by centrifugation at 13000  rpm for 3  min .
supernatant ( cytosolic fraction ) and pellets ( hm fraction ) were analyzed by western blotting or further processed .
for preparation of mitochondria enriched fractions , jurkat cells were resuspended in buffer ( 10  mm nacl , 1.5  mm mgcl2 , 10  mm tris - hcl ) and left for 10  min on ice .
hypotonic swelling was stopped by addition of buffer ( 525  mm mannitol , 175  mm sucrose , 12.5  mm tris - hcl ph  7.5 , 2.5  mm edta ) and cells ruptured by dounce homogenization . after clearing lysates at 2700  rpm for 10  min
, mitochondria enriched fractions were pelleted at 13 000  rpm for 15  min and washed in x - link buffer.sirna transfectionsjurkat cells were transfected using nucleofection ( amaxa , kln , germany ) , following the manufacturer s instructions with a single bak sirna gcuuuagcaagugugcacutt , ambion , paisley uk ) or a pool of sirnas ( cgacaucaaccgacgcuautt , uaugaguacuucaccaagatt , gacggcagcucgccaucautt , aaucaugacucccaagggutt , thermo fischer ) , at a final concentration of 75  nm .
a nonspecific control pool , containing four pooled nonspecific sirna duplexes , was used as a negative control . for bim knockdown three single sirnas ( bim-1 , gaccgagaagguagacaautt ; bim-2 , gcaaccuucugauguaagutt ; bim-3 , ccucccuacagacagagcctt [ ambion ] )
were transfected independently into hek293 t cells using interferin ( polyplus ) according to the manufacturer s protocol . after 24  hr
, cells were reconstituted with bcl - xl : bak complexes . after a further 48  hr ( bak ) or 72  hr ( bim ) , knockdown efficiency
was determined by sds - page and the cells further processed.immunoprecipitationsfor immunoprecipitation , cells were lysed in lysis buffer ( 20  mm tris - hcl ph 7.4 , 135  mm nacl , 1.5  mm mgcl2 , 1  mm egta , 10% glycerol ) containing 1% chaps or 1% triton x-100 and supplemented with protease inhibitor cocktail ( roche diagnostics , switzerland ) and incubated on ice for 20  min .
lysates were pre - cleared by centrifugation at 13000  rpm for 15  min .
rabbit bak ab ( upstate biotechnology ) , mouse bak ab-1 antibody ( calbiochem ) , rabbit bcl - xl antibody ( bd biosciences ) , hamster bcl-2 antibody ( bd bioscience ) , rabbit bim antibody ( cell signaling technologies ) or rabbit mcl-1 antibody ( epitomics , burlingame ca ) were crosslinked to protein a - dynabeads using 20  mm dimethylpimelimidate ( fluka biochemika , switzerland ) .
crosslinked antibodies were incubated with 0.5 - 1  mg protein for 12  hr at 4c .
beads were washed with lysis buffer supplemented with tween-20 before elution in sodium dodecyl sulfate ( sds ) loading buffer and western blotting .
immunoprecipitations against strep - tag ii or flag - tag were carried out using streptavidin coated agarose beads ( iba , goettingen germany ) or anti - flag m2 crosslinked agarose beads ( sigma aldrich , dorset uk).crosslinking of bak oligomers using egsmitochondria enriched fractions from jurkat cells or digitonin generated heavy membrane fractions from mefs were solubilized in x - link buffer and incubated with cuphe ( 20  mm 1,10 phenanthroline with 300  mm cuso4 ) for 30  min on ice .
heavy membrane fractions were incubated with egs ( 5  m ) for 30  min on ice .
bak complexes were then analyzed by sds-page.statisticsall statistics were performed using spss , and significant differences were determined using a one - way anova with a fisher s least significant difference post hoc analysis .
cell lines were obtained from the european collection of animal cell cultures ( ecacc ; porton down , uk ) except where indicated .
jurkat cells ( clone e6.1 ) were maintained in roswell park memorial institute ( rpmi ) medium containing 10% fetal calf serum ( fcs ) and 5% glutamax ( life technologies inc .
paisley uk ) , human embryonic kidney fibroblasts ( hek293 t ) and mefs deficient in bax and bak were cultured in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% ( fcs ) and 5% glutamax. the intrinsic apoptotic pathway was induced by treatment with abt-737 ( abbott laboratories , illinois , usa ) with the concentration used dependent on the cell type ( 3 - 30  m ) .
apoptosis was measured using annexin v and propidium iodide ( pi ) staining . for non - adherent cells , annexin buffer ( 10  mm hepes / naoh ( ph 7.4 ) , 150  mm nacl ,
5  mm kcl , 1  mm mgcl2 , 1.8  mm cacl2 ) supplemented with annexin v - fitc was added to the cells .
after incubation for 10  min , propidium iodide was added and cells analyzed on a facscan flow cytometer using cellquest software ( becton dickinson , oxford uk ) . for adherent lines , both detached and trypsinized cells were resuspended in fresh medium and left to recover prior to staining .
tmre ( 100  nm ) was added to medium and cells incubated at 37c for 10  min in the dark prior to analysis by flow cytometry .
for intracellular ab-1 staining , cells were fixed with 2% paraformaldehyde at room temperature for 10  min , washed with pbs and resuspended in permeabilization buffer ( 0.1% saponin and 0.5% bsa in pbs ) containing bak ab-1 antibody ( calbiochem , nottingham , uk ) ( 1:100 dilution ) .
after incubation for 1  hr at 4c , cells were washed and incubated with goat anti - mouse igg - alexa fluor 488 ( invitrogen ) diluted 1:100 into permeabilization buffer .
for limited trypsin proteolysis , mitochondria enriched fractions were resuspended in x - link buffer ( 100  mm sucrose , 20  mm hepes - koh ph 7.4 , 2.5  mm mgcl2 , 50  mm kcl ) and 125  g trypsin/50  g protein was added . following incubation for 20  min on ice trypsin
was inhibited by addition of 4  mm pefabloc sc ( roche diagnostics , basel , switzerland ) .
after 24  hr , cells were fixed with 4% ( v / v ) paraformaldehyde and permeabilized with 0.5% saponin in pbs .
cells were then incubated with anti - cytochrome c and ab-1 followed by the appropriate fluorophore - conjugated secondary antibodies ( alexa 547 for cytochrome c and alexa 488 for ab-1 ) and hoechst 33342 .
coverslips were then mounted on glass slides and subjected to confocal microscopy on a zeiss lsm510 .
heavy membrane ( hm ) fractions were generated by resuspending in mitochondrial isolation buffer ( 250  mm sucrose , 20  mm hepes , ph 7.4 , 5  mm mgcl2 and 10  mm kcl ) containing 0.01% digitonin .
cells were left on ice followed by centrifugation at 13000  rpm for 3  min .
supernatant ( cytosolic fraction ) and pellets ( hm fraction ) were analyzed by western blotting or further processed .
for preparation of mitochondria enriched fractions , jurkat cells were resuspended in buffer ( 10  mm nacl , 1.5  mm mgcl2 , 10  mm tris - hcl ) and left for 10  min on ice .
hypotonic swelling was stopped by addition of buffer ( 525  mm mannitol , 175  mm sucrose , 12.5  mm tris - hcl ph  7.5 , 2.5  mm edta ) and cells ruptured by dounce homogenization .
after clearing lysates at 2700  rpm for 10  min , mitochondria enriched fractions were pelleted at 13 000  rpm for 15  min and washed in x - link buffer .
jurkat cells were transfected using nucleofection ( amaxa , kln , germany ) , following the manufacturer s instructions with a single bak sirna gcuuuagcaagugugcacutt , ambion , paisley uk ) or a pool of sirnas ( cgacaucaaccgacgcuautt , uaugaguacuucaccaagatt , gacggcagcucgccaucautt , aaucaugacucccaagggutt , thermo fischer ) , at a final concentration of 75  nm .
a nonspecific control pool , containing four pooled nonspecific sirna duplexes , was used as a negative control . for bim knockdown three single sirnas ( bim-1 , gaccgagaagguagacaautt ; bim-2 , gcaaccuucugauguaagutt ; bim-3 , ccucccuacagacagagcctt [ ambion ] ) were transfected independently into hek293 t cells using interferin ( polyplus ) according to the manufacturer s protocol .
after 24  hr , cells were reconstituted with bcl - xl : bak complexes . after a further 48  hr ( bak ) or 72  hr ( bim ) , knockdown efficiency
, cells were lysed in lysis buffer ( 20  mm tris - hcl ph 7.4 , 135  mm nacl , 1.5  mm mgcl2 , 1  mm egta , 10% glycerol ) containing 1% chaps or 1% triton x-100 and supplemented with protease inhibitor cocktail ( roche diagnostics , switzerland ) and incubated on ice for 20  min .
lysates were pre - cleared by centrifugation at 13000  rpm for 15  min .
rabbit bak ab ( upstate biotechnology ) , mouse bak ab-1 antibody ( calbiochem ) , rabbit bcl - xl antibody ( bd biosciences ) , hamster bcl-2 antibody ( bd bioscience ) , rabbit bim antibody ( cell signaling technologies ) or rabbit mcl-1 antibody ( epitomics , burlingame ca ) were crosslinked to protein a - dynabeads using 20  mm dimethylpimelimidate ( fluka biochemika , switzerland ) .
crosslinked antibodies were incubated with 0.5 - 1  mg protein for 12  hr at 4c .
beads were washed with lysis buffer supplemented with tween-20 before elution in sodium dodecyl sulfate ( sds ) loading buffer and western blotting .
immunoprecipitations against strep - tag ii or flag - tag were carried out using streptavidin coated agarose beads ( iba , goettingen germany ) or anti - flag m2 crosslinked agarose beads ( sigma aldrich , dorset uk ) .
mitochondria enriched fractions from jurkat cells or digitonin generated heavy membrane fractions from mefs were solubilized in x - link buffer and incubated with cuphe ( 20  mm 1,10 phenanthroline with 300  mm cuso4 ) for 30  min on ice .
heavy membrane fractions were incubated with egs ( 5  m ) for 30  min on ice .
all statistics were performed using spss , and significant differences were determined using a one - way anova with a fisher s least significant difference post hoc analysis .
 summarybak activation represents a key step during apoptosis , but how it converts into a mitochondria - permeabilizing pore remains unclear . by further delineating the structural rearrangements involved 
 , we reveal that bak activation progresses through a series of independent steps : bh3-domain exposure , n - terminal change , oligomerization , and membrane insertion . 
 employing a  bcl - xl - addiction  model , we show that neutralization of bcl - xl by the bh3 mimetic abt-737 resulted in death only when cells were reconstituted with bcl - xl : bak , but not bcl-2/ bcl - xl : bim complexes . 
 although this resembles the indirect model , release of bak from bcl - xl did not result in spontaneous adoption of the pore conformation . 
 commitment to apoptosis required association of the direct activator bim with oligomeric bak promoting its conversion to a membrane - inserted pore . 
 the sequential nature of this cascade provides multiple opportunities for other bcl-2 proteins to interfere with or promote bak activation and unites aspects of the indirect and direct activation models . 
Introduction
Results
Discussion
Experimental Procedures