BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate

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1 H3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation o ax Florian Willing 1, rnim Weber 1, Stephanie Pottho, F.-Nora Vögtle, hris Meisinger, Stean. Paschen and Georg Häcker dditional Footnotes 1 share irst authorship Supplementary Inormation ontains Supplemental Figures S1-S11 1

2 Figure S1 Hydropathicity average ad id ik im m... ELRRMSDEFEGSFKGLPRPKSGQMRQSGWRIIQWDRNLGKGGSPSQ-... DMENDKMLIMMLLKKVSHPSLLRDVFHVNFINQNLFSYVRNLVRNEMD-... RSLIRSLNLRENIWRVLPGWVSPDQDPGQLFPMVLLVFLLLGGWYLQLQ-... PEIRIQELRRIGDEFNEYRRVFNDYREEDHPQMVILQLLRFIFRLVWRRH-... EVQIRKLQIDQFHRLHQQHQQNRDRWWQVFLFLQNLLNRQENREGVGPW Noxa... QMPGKSQKSRMRSPSPRVPDLKDEQLRRIGDKVNLRQKLLNLISKLFNLV Puma... QLRRMDDLNQYERRRQEEQHRHRPSPWRVMYNLFMGLLPLPRDPGPEMEPN- = putative transmembrane domain = -terminal sequence used or GFP usion = -terminal sequence deleted in H3-only protein Figure S1 Sequence analysis o pro-apoptotic H3-only proteins () Identiication o tail-anchor sequences within H3-only proteins. he last 50 amino acid residues o the H3-only proteins ad, id, ik, im, m, Noxa and Puma are shown. Putative transmembrane domains identiied by in silico prediction using various computer programs are boxed. Positive-charged amino acid residues are indicated in blue (partially essential or mitochondrial targeting and/or as potential trypsin cleavage sites). he average hydropathicity or the predicted transmembrane domain was calculated according to the scale o Kyte and Doolittle (EXPasy, ProtParam tool). Putative tail-anchor sequences used or GFP-usion proteins (see Figures 2, S2, S4, S5, S7) o each H3-only protein are indicated by the bracket symbol. () Schematic and not-to scale outline o H3-only protein mutants used. GFP-H3-only protein-, usion protein o GFP and the indicated tail-anchor domains o H3-only proteins (Fig. S1), murine Fis1 (aa , see Fig. S6) or yeast om5 (aa 23-50, yom5, Fig S4); H3-only protein, the tail-anchor domains o H3-only proteins were deleted as indicated by the red brackets in Fig. S1. Point and deletion mutants o im S and are shown.

Figure S2 P S P S P S P S GFP-im- L P S P S im L im S L / P S

Puma Puma S P S P S P S P F GFP-Puma- L P S P S Puma Puma Puma

Puma are mitochondrial tail-anchored proteins Import o in

S1) (), or the -terminus o im used to the -terminus o GFP (),

Puma used to the -terminus o GFP (F) into mitochondria.

isolated rom Saccharomyces cerevisiae cells.

One part was let untreated (-), another one was treated with

nother part was subjected to hypotonic swelling and trypsin

he two last parts were subjected to without or with

containing integral membrane proteins) and supernatant

3 Figure S2 P S P S P S P S GFP-im- L P S P S im L im S L / P S P S L / P S P S D id L / P S P S im L im S E PumaΔ L / P S Puma Puma S P S P S P S P F GFP-Puma- L P S P S Puma Puma Puma Puma Figure S2 In vitro evidence that H3-only proteins im and Puma are mitochondrial tail-anchored proteins Import o in vitro translated and (lacking the -terminal tail-anchor, Fig. S1) (), or the -terminus o im used to the -terminus o GFP (), im L or im S (), id (D), Puma or Puma (E) or the -terminus o Puma used to the -terminus o GFP (F) into mitochondria. Radiolabeled proteins were incubated with mitochondria isolated rom Saccharomyces cerevisiae cells. Subsequently, the samples were divided. One part was let untreated (-), another one was treated with 100 μg/ml trypsin () or 30 min on ice. nother part was subjected to hypotonic swelling and trypsin treatment (/). he two last parts were subjected to without or with pre-treatment with trypsin resulting in pellet ractions (P; containing integral membrane proteins) and supernatant ractions (S; containing soluble and peripherally attached proteins). For comparison, 10% o the total input o radiolabeled precursors was included (L). Mitochondria were re-isolated and import was analyzed by SDS-PGE and autoradiography. F, proteolytic ragments o im. Molecular weight markers are indicated. In some cases some lanes were digitally removed.

Figure S3 / P S P S / P S P S cyt c Su9-DHFR p L PK 5 100 5 100 P S D Fis1 L P S Su9-DHFR m Figure S3 Localisation o control proteins with known submitochondrial localisation (, ) he membrane o the

4 Figure S3 / P S P S / P S P S cyt c Su9-DHFR p L PK P S D Fis1 L P S Su9-DHFR m Figure S3 Localisation o control proteins with known submitochondrial localisation (, ) he membrane o the import shown in Fig. 1 was probed with antibodies against mitochondrial DP/P carrier (, localized at the inner mitochondrial membrane, ) and cytochrome c (intermembrane space but released upon disruption, ). (), Import o in vitro translated Su9-DHFR into isolated yeast mitochondria. Su-9DHFR is a mitochondrial matrix protein consisting o residues 1-69 o Neurospora crassa mitochondrial Pase subunit 9 (Su9, containing the matrix targeting signal) used to ull-length mouse dihydroolate reductase; this protein is imported into the mitochondrial matrix. Radiolabeled protein was incubated or 10 min at 25 with mitochondria isolated rom yeast cells. Subsequently, the sample was divided. One part was treated with 5 or 100 μg/ml trypsin () or 5 and 100 μg/ml proteinase K or 30 min on ice, another one was let untreated. he last part was subjected to (P; containing integral membrane proteins) and supernatant ractions (S; containing soluble and peripherally attached proteins). For comparison, 10% o the total input o radiolabeled precursors was included (L). Mitochondria were re-isolated and import was analyzed by SDS-PGE and autoradiography. p, Su9-DHFR precursor; m, mature orm o Su9-DHFR ater import and cleavage via the matrix-localized processing peptidase. (D) lkaline o in vitro translated and imported mfis1 (located in the outer mitochondria membrane) into yeast mitochondria. Localisation appears similar to im (Fig. 1, Fig. S2).

Figure S4 GFP L P S P S GFP-om5- L P S P S Figure S4 In vitro import o GFP or GFP-om5- into isolated yeast mitochondria Import o in vitro translated GFP () or GFP-om5- (; GFP used N-terminally to the

Radiolabeled proteins were incubated or 10 min at 25 with mitochondria isolated rom yeast cells. Subsequently, the samples were divided.

5 Figure S4 GFP L P S P S GFP-om5- L P S P S Figure S4 In vitro import o GFP or GFP-om5- into isolated yeast mitochondria Import o in vitro translated GFP () or GFP-om5- (; GFP used N-terminally to the tail-anchor domain o yeast om5 (aa 23-50)) into isolated yeast mitochondria. om5 is a tail-anchor protein o the mitochondrial outer membrane. Radiolabeled proteins were incubated or 10 min at 25 with mitochondria isolated rom yeast cells. Subsequently, the samples were divided. One part was treated with 100 μg/ml trypsin () or 30 min on ice. he two last parts were subjected to without or with pre-treatment with trypsin resulting in pellet ractions (P; containing integral membrane proteins) and supernatant ractions (S; containing soluble and peripherally attached proteins). For comparison, 10% o the total input o radiolabeled precursors was included (L). Mitochondria were re-isolated and import was analyzed by SDS-PGE and autoradiography. F, proteolytic ragment o om5. Molecular weight markers are indicated. Western blots are representative o two independently perormed experiments.

Figure S5 Noxa L P S P S GFP-Noxa- L P S P S NoxaΔ / P S.

proteins Import o radiolabeled proteins into isolated yeast mitochondria and analysis o

() Import o GFP-Noxa- (the putative tail-anchor domain o Noxa used -terminally to GFP, Fig.

6 Figure S5 Noxa L P S P S GFP-Noxa- L P S P S NoxaΔ / P S.4 D m L / P S P S E ik PK L PK P S P S Figure S5 Mitochondrial in vitro import o H3-only proteins Import o radiolabeled proteins into isolated yeast mitochondria and analysis o membrane association as described in Figure S4. () Import o Noxa. () Import o GFP-Noxa- (the putative tail-anchor domain o Noxa used -terminally to GFP, Fig. S1). (), import o Noxa lacking the -terminal tail-anchor, Fig S1). Import o m (D) or ik (E)., proteolytic ragments o ik., trypsin treatment; PK, proteinase k treatment; /, swelling ollowed by trypsin treatment. P, S designate pellet/supernatant ractions ollowing. Western blots are representative o two independently perormed experiments.

Subcellular localization o controls GFP,

analyzed by conocal microscopy s control

tail-anchor domain o Fis used -terminally

S1); ull length im L ΔΔ (a mutant unable

Mitochondria were identiied by Mitoracker

7 Figure S6 GFP Mitoracker overlay inset Matrix-GFP GFP-im GFP-Fis1- GFP Figure S6 Subcellular localization o controls GFP, GFP-Fis-, GFP-im L ΔΔ, or Matrix-GFP as analyzed by conocal microscopy s control proteins were used: GFP; the -terminal tail-anchor domain o Fis used -terminally to GFP (see Fig. S1); ull length im L ΔΔ (a mutant unable to bind to cl-2 amily members) used -terminally to GFP; a chimeric protein consisting o GFP and an N-terminal usion with the mitochondrial matrix targeting signal o ornithine carbamoyl-transerase (matrix-gfp). Localization o GFP-usion proteins (green) upon transient transection o HeLa cells was determined by conocal microscopy. Mitochondria were identiied by Mitoracker staining (red; staining o mitochondrial matrix). Various overlays and details (boxed) are shown. ars, 10 μm and 1 μm or the inset.

8 Figure S7 GFP ER-DsRed overlay inset GFP-ik- GFP ER-DsRed overlay inset GFP-ad- GFP-id- GFP-Noxa- GFP-Puma- GFP-Fis1- GFP-im GFP-im GFP-im- Figure S7 Subcellular localization o H3-only-GFP usions as analyzed by conocal microscopy he -terminus o im (GFP-im-), wt im L (GFP-im) or a im L mutant (GFP-imΔΔ, with inactive H3-domain), and -termini rom other indicated proteins (see Fig. S1) were used to the -terminus o GFP and the constructs were transiently expressed in HeLa cells. Subcellular localization o GFP-usion proteins (green) was analyzed by microscopy. Endoplasmic reticulum (ER) was stained by cotransecting a DsRed2 variant containing the ER targeting signal o calreticulin and an ER retention signal (ER-DsRed). Overlays and details (boxed) are shown. he bar gives a length o 10 μm or 1 μm (inset). Over 90 % o GFP-positive cells showed the pattern described here.

Figure S8 aspase-8 M M M M M M aspase-8 M M M M M M oxiv oxiv im W α: Hsp60 ubulin 25 con EL ELΔΔ ELΔ con + M M M id endogenous * id tid EL + D aspase-8 oxiv - + tid - + Puma - + hnoxa M M M M M M 10

9 Figure S8 aspase-8 M M M M M M aspase-8 M M M M M M oxiv oxiv im W α: Hsp60 ubulin 25 con EL ELΔΔ ELΔ con + M M M id endogenous * id tid EL + D aspase-8 oxiv - + tid - + Puma - + hnoxa M M M M M M 10 tid tid id tid im L DKO - + Imi 1205 WM35 Sbcl2 Haa E et [h] aspase-8 mitochondria cytosol F aspase-8 M P S oxiv oxiv cyt c cyt c him S ad mut2 im S Figure S8 Subcellular localization o wild-type and mutant H3-only proteins in mammalian cells (-) ax -/- /bak -/- DKO MEFs were retrovirally transduced using empty vector control (con) or pmig-ires-gfp vectors coding or murine ull lenght, ΔΔ(L150/I153), Δ (all three are mutants where the splice site has been deleted to prevent generation o im L and im S ) (), tid, Puma, or human Noxa (hnoxa) () or tid, tidδ, or idδ (). Mitochondrial ractions (M) and soluble ractions ollowing centriugation at 100,000 x g () were separated by SDS-PGE and detected by Western blotting using the appropriate antibodies. aspase-8 or tubulin (cytosol) and oxiv or Hsp60 (mitochondria) served as marker proteins. Where indicated 5 μm -737 (or DMSO as control solvent (-)) was added 24 h beore isolation o mitochondria. Note that only cell populations expressing >90% GFP (rom the pmig-ires-gfp vectors which code or the H3-only proteins indicated) were used. *crossreactive band o unknown origin. (D) o compare subcellular localisation o im in various human cell lines, mitochondria (M) and the corresponding cytosolic ractions () were analyzed by SDS-PGE and Western blotting. aspase-8 (against mouse (let blot), and human (right)) and oxiv served as marker or the ractions. Note that 5 μg o mitochondria was loaded rom bax -/- /bak -/- DKO MEFs (DKO ); or the human keratinocyte cell line (Haa) and the three human melanoma cell lines (1205, WM35 and Sbcl2) 35 μg mitochondria and equal volumes o cytosol was used. Haa cells were incubated with or without 50 μg/ml imiquimod (Imi) or 24 h in the presence o zvd-mk (75 μm) beore mitochondria were isolated. (E) ime-dependent (1-6 h) induction o mouse im S in the tetracycline (tet)-inducible mouse melanoma cell line 16-im S (33) irst results in translocation o im S to mitochondria and at later time points the accumulation in the cytosol. o inhibit im S -induced apoptosis 75 μm zvd-mk was added 30 min beore addition o tet. (F) Isolated mitochondria (M) and the corresponding cytosolic ractions () rom ax -/- /bak -/- DKO MEFs retrovirally transduced with pmig-ires-gfp coding or himsady110i/s118g (himsad mut2 ; amino acid substitutions in the wild type ad-h3 domain at the same position as in the himsad chimera (mut2) enable the ad to bind to all cl-2 amily proteins (26)) were analyzed by SDS-PGE and Western blotting or the presence o the indicated proteins. ter (0.1 M sodium carbonate, ph 11.5) membrane integrated proteins (like himsad mut2 ) stay in the pellet ractions (P) whereas soluble or associated proteins appear in the soluble ractions (S). ytochrome c (cyt c) served as a positive control or released during disruption o mitochondria); oxiv as an integral membrane protein is ound in the resistant mitochondrial raction (P).

10 Figure S9 ax [nm] P S P S P S P S P S 500 P S P S P S P S cyt c ax him S ad mut2 con him S bax -/- /bak -/- -DKO MEFs expressing: con hnoxa Mcl-1 hnoxa ubulin supernatant (S1) mitochondria (+/-PK) (bax -/- /bak -/- -DKO MEFs) +/- in vitro translated protein 30 min 30 x10,000g 1x wash & incubation o mitoch. +/- recombinant ax (30 min, 30 ) x10,000g supernatant (S2) 1x wash mitoch. pellet (P) Figure S9 ax-induced cytochrome c release rom mitochondria expressing human im (him S ) but not mutant im S () Mitochondrial ractions rom bax -/- /bak -/- DKO MEFs were directly tested or their ability to activate ax to release cytochrome c (cyt c). Recombinant human ax protein was incubated (30 min 30 ) at the indicated concentrations with mitochondria (30 μg) rom cells expressing himsad mut2 or human im S (him S ), (see Fig. 3, S8). Reactions were split into mitochondria (P) and supernatant (S) by centriugation and analyzed or cytochrome c-content. con, empty vector control. Western blots are representative o at least two independently perormed experiments. () Overexpression o human Noxa reduces levels o Mcl-1 Representative Western blot o cell lysates rom bax -/- /bak -/- DKO MEFs retrovirally transduced with pmig-h-hnoxa (hnoxa) or empty vector control (con) showing reduced levels o endogenes Mcl-1. ubulin was used as a loading control. () Experimental setup or the 3xH-im import and subsequent release o cyt c by the activation o recombinant ax using isolated bax -/- /bak -/- DKO MEF mitochondria pretreated with or without proteinase K (PK, see Fig. 5). PK treatment and im import were perormed in M-ED buer; ax aktivation was done in Kl buer.

Figure S10 W ax ax/ ax/ Δ ax/ ΔΔ ax/ GFP ax uninduced (+tet) ax induced (-tet) Figure S10 accelerates the cell death-inducing eect o ax in yeast Yeast strains containing ax, ax/, ax/, ax/ or ax/gfp

ells were then diluted in 10-old increments and spotted on SD plates containing 2% glycerol with and without tetracycline (1 μg/ml) to induce ax protein expression.

11 Figure S10 W ax ax/ ax/ Δ ax/ ΔΔ ax/ GFP ax uninduced (+tet) ax induced (-tet) Figure S10 accelerates the cell death-inducing eect o ax in yeast Yeast strains containing ax, ax/, ax/, ax/ or ax/gfp (as control) were grown to log phase in synthetic medium containing 1 μg/ml tetracycline lacking uracil and leucin (SD-Ura/-Leu), washed 3 times and diluted in distilled water to an OD600 o 0.5. ells were then diluted in 10-old increments and spotted on SD plates containing 2% glycerol with and without tetracycline (1 μg/ml) to induce ax protein expression. ter the spotting the cells were incubated or 4 days at 30 and imaged with a D camera. W, wild-type yeast strain. ll expressed proteins (and variants, see Figure S1) including ax were rom mouse. Pictures show one representative o three independent perormed experiments.

12 Figure S11 inactive ax diusion transient interaction + conormational change inactive ax diusion N cytosol active ax N cytosol N cytosol ax activation OMM OMM ax insertion active ax OMM H3-only protein IMS H3-only protein IMS H3-only protein IMS Figure S11 Model o ax recruitment and activation by tail-anchored H3-only proteins ytosolic, inactive ax reaches the mitochondrial surace by passive diusion (). transient interaction between certain H3-only proteins (tid/im/pum), which are inserted into the outer mitochondrial membrane (OMM) via their -terminal tail-anchor, triggers a conormational change o ax (). his conormational change allows or the insertion o ax into the OMM, and the process is repeated with the next ax molecule until enough ax is inserted into the OMM to permit oligomerisation and cytochrome c release rom the intermembrane space (IMS) (). he localisation o H3-only proteins as tail-anchored OMM proteins is required to achieve an active conormation o the H3-only protein and/or to enable transiently activated ax to insert into the OMM beore it reolds into the inactive conormation.

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