SUPPLEMENTARY INFORMATION

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1 DOI: /n2347 H.s_C MS--TLF M.m_C11 1 M RKLRPREVKCIGQNQRARAMS--TLF X.l_C MS--TLF D.r_C MS--ALF D.m_C MSMIPFF S.p_APC MS----F S._Mnd MARALRDISLFNDIRKDQNSAGAKHERYNMRDLRSKKNQHVNGIDDYEDDSLDRFIRRKKSRVVKYI 67 H.s_C11 6 PSL--FPRVTETLWFNLDRPCV-EETELQQ--QEQQHQAWLQSIAEKD-NNLVPIG--KPASE M.m_C11 26 PSL--FPRVTETLWFNLDRPCV-EETELQQ--QEQQHQAWLQSIAEKD-NNLVPIG--KPASE X.l_C11 6 PSL--FPQVTDSLWFNLDRPCV-DENELQQ--QEQQHQAWLLSIAEKD-TGLVPIG--KPASE D.r_C11 6 PSL--FPRVTESLWFDLDRPCV-DEAELNQ--QEQQHQTWLQSIVEKD-NNLMPIG--RPIFE D.m_C11 8 PTL--KVSVANRYWLDMAPASVNEESQTRR--YEDERSNWRESLKTAG-TDLQPLG--KMLTI S.p_APC 4 MSL--MANTAHQLWYPTSFPSM---KELEK--EEVRLETQELAIKQFGFRTIRPIGLNRSMQE S._Mnd2 6 8 PSLSAYNVFNEFPYYPTSASQLLDGKLDEFLMLSEQYKSRLPKIRKLGWNRFKPIGINKTMYELEMLRSRARAQNAEGNNEED 0 H.s_C HYDDEEEE DDEDDEDSEEDSED D 83 M.m_C HYDDEEEE DDEDDEDSEEDSED D 103 X.l_C PYDEEEEE DDEDDEDSEEDSED D 83 D.r_C TFDEEEEE DDEDEEDSEEDSED D 83 D.m_C P---GIET DDEDANDDSEDTDSH D 84 S.p_APC QLDLEEQE REEANQDTELDEEELSGSFPEEG 86 S._Mnd2 1 EDFRQHDSREEDPRNNGSIGRVILPHILQENEEYDTGEGVTGLHSMPNDSMAILANNSANNSQNEEVSEEDEISYDYDAEFDH 2 H.s_C11 84 E----DMQDMDEMNDYNESPDDG EVNEVD--MEGNEQDQDQWMI 1 M.m_C1 104 E----DMQDMDEMNDYNESPDDG EVNEVD--MEGNEQDQDQWMI 1 X.l_C11 84 E----DMQDMDEMNDYNESPDDG EI-EAD--MEGAEQDQDQWMI 1 D.r_C11 84 E----DMQDMDDMNIYNEFPDDG EINEVD--MEGADQDQDQWMI 1 D.m _C E----EDDETND----RVIPVTQ DFYSADDIQMNDETSPTAP S.p_APC 8 7 G----EMDDMDG---DNEEVEDH DIEEVD--LDADITNADASEF 125 S._Mnd2 232 VVDEDDNEEGEVPGEGVEGIEVQRERIVPDDLLMRPTSLSRSLQQFVEEAHHLDRNPYDIDSDNDGEDSKVELDMNPDFEDDV 4 Id.% E vlue H.s_C M.m_C X.l_C D.r_C D.m_C S.p_APC 1 26 YEDI---SEYGFQN S._Mnd2 3 G REHDYNSEYSQEPTSYGGITPDLASNWRNWTRERITSLDELMERRARQQRGQD e -84 2e -43 3e -48 3e reominnt APC MW (kd) 200 APC 6 α APC Figure S1 ClustlW lignment of C11orf51/hAPC nd hrteristion of the nti-apc ntiody. () Alignment of humn C11orf51 with Shizoshromyes pome APC 6, Shromyes erevise Mnd2 7 nd other model orgnisms (Mus musulus, Xenopus levis, Dnio rerio, Drosophil melnogster). Residues tht re present in ll seven speies re highlighted in lk, residues present in four or more speies re shded in drk grey nd similr residues in light grey. Expeted vlue (E) ws lulted using BLASTP nd the indited orgnisms. For Mnd2 only E versus S. pome Ap is shown. () Asynhronously growing HeL ells were treted with the indited sirnas t 50 nm for 85h. Cells were lysed diretly in SDS-smple uffer nd nlysed y immunolotting with n nti- APC ntiody. 10 ng of full-length reominnt APC ws used s positive ontrol. Moleulr mss mrkers on the right. Results representtive of two experiments. 1

2 α APC8 α APC α flg input tr IP APC4 IP flg IP tetryline APC-3xflg endo. APC LC input tr IP APC3 IP unound sirna: α APC3 α APC6/8 α APC α α tuulin GAPDH APC3 APC6 APC8 GAPDH GAPDH APC3 APC6 APC8 GAPDH APC3 APC6 APC8 APC6 (72) APC8 (68) si-apc6: : APC10 APC3 APC7 APC11 APC6 APC8 AB APC5 APC4 APC2 APC1 M r time fter relese (h) α Cylin B1 α Cylin A2 α α phospho-h3 α α tin Figure S2 APC requires APC8 for inorportion into the APC/C nd APC depletion delys Cylin B1 ut not Cylin A degrdtion. () HeL ells were treted with the indited sirnas for 72h nd rrested in mitosis with DMA. APC-3xflg ws indued with tetryline 72h efore the experiment. Immunolot nlysis of nti-apc4 nd nti-flg immunopreipittes tht were nlysed with the indited ntiodies. LC denotes the light-hin of nti-apc4 nd nti-flg ntiodies. Moleulr mss mrkers on the right. Results representtive of three experiments. () Immunopreipittion of the APC3, APC7 nd APC10 suomplex of the experiment shown in Fig. 1d tht ws preipitted y nti-apc3 ntiodies (see rtoon), nd nlysed with the indited ntiodies. Moleulr mss mrkers on the right. Results representtive of three experiments. () HeL ells treted with the indited sirna oligonuleotides for 85h were relesed from doule thymidine lok, olleted t the indited time-points nd nlysed y immunolotting with the indited ntiodies. Moleulr mss mrkers on the right. Results representtive of two experiments. 2

Mnsfeld et l, Supplementry Figure 3 normlised fluoresene Cylin B1-Venus degrdtion oligo 1 oligo 2 oligo 3 oligo 4 oligo 6 0 10 20 30 40 50 60 70 80 90 time reltive to NEBD (min) NEBD to nphse n= 118

3 Mnsfeld et l, Supplementry Figure 3 normlised fluoresene Cylin B1-Venus degrdtion oligo 1 oligo 2 oligo 3 oligo 4 oligo time reltive to NEBD (min) NEBD to nphse n= oligo 4 oligo 4 + APC-IRES-mRUBY Figure S3 APC depletion delys nphse. () RPE1-Cylin B1-Venus ells were treted for 85h with 50 nm of the indited sirna oligonuleotides efore nlysis y time-lpse mirosopy. Montge of representtive imges showing the prolonged metphse rrest in APC-depleted ells. Sle r, 10 mm, (NEBD = 0 min, phse=phse-ontrst mirosopy). () Single ell destrution ssys of synhronously growing RPE1-Cylin B1-Venus ells treted s in () using five different oligonuleotides trgeting APC. Fluoresene intensities were normlised to NEBD. Destrution urves show the men nd s.d. of 25 ells per oligonuleotide from three experiments. () For resue experiments APC-siRNA oligonuleotide 4 trgeting the 3 UTR of APC ws used. Asynhronously growing RPE1 ells with tetryline-induile APC were treted with 50 nm of the indited sirna oligonuleotides for 85h efore the timing from NEBD to nphse ws determined y phse-ontrst mirosopy. Expression of untgged APC nd mruy trnslted from the sme mrna using n IRES (internl riosome entry site) element ws indued y the ddition of tetryline 72h efore nlysis. Only ells expressing detetle levels of mruy were nlysed. Stter dot lots show the men (red line) of the indited numer of ells from two experiments. 3

4 NEBD Cylin B1-Venus (0.5 μeversine) normlised fluoresene + (n=33) si-apc10 + (n=25) + (n=25) si-apc10 + (n=25) time reltive to NEBD (min) tetryline α α α input tr IP APC3 IP unound α APC APC-3xflg endo. APC rel. inding to the APC/C ( +tet = 1) Binding to the APC/C + tet tet perentge slippge (umulted numer) mitoti slippge (5 μm txol) si-p omet Figure S4 Depletion of APC nd APC10 does not hve n dditive effet on APC/C tivity in vivo, etopi expression of sirna-resistnt APC resues the umultion of MCCs, nd depleting APC or p omet prevents mitoti slippge. () Single ell destrution ssys of synhronously growing RPE1-Cylin B1-Venus ells treted for 85h with the indited sirna oligonuleotides. To mesure APC/C-tivity independently of the SAC, 0.5 meversine ws dded to the medium prior to filming. Destrution urves show men nd s.d. of the indited numer of ells from two experiments. () HeL ells ontining tetryline-induile APC-3xflg were treted for 85h with the indited sirnas nd rrested in mitosis with DMA. APC/C ws purified with nti-apc3 ntiodies nd preipittes were nlysed y immunolotting with the indited ntiodies. For quntifition the mount of MCCs in non-indued (-tet) nti-apc3 preipittes from -treted ells ws normlised to the mount in tet-indued (+tet) -treted ells. Brs indite men nd s.e.m from three experiments. () Mitoti slippge of RPE1-Cylin B1-Venus ells in the presene of 5 µm txol ws nlysed y time-lpse mirosopy (si- GAPDH, n=91; n=; si-p omet n=42) from 2 experiments. 4

5 APC/C MCC frtion α α α V kd 670 kd 8 kd input (1/10) 117 rel. protein mount APC frtions frtion α α α input (1/10) APC/C MCC V kd 670 kd 8 kd 117 rel. protein mount APC frtions frtion α α α V kd 670 kd 8 kd input (1/10) si-p omet APC/C MCC 117 rel. protein mount si-p omet APC frtions Figure S5 Size-exlusion hromtogrphy of GADPH-, APC- nd p omet - depleted ells. HeL ell extrts from prometphse-rrested ells depleted of GAPDH (), APC () nd p omet () were nlysed y size-exlusion hromtogrphy. The frtions were proed with the indited ntiodies nd nlysed y quntittive immunolotting. Moleulr mss mrkers on the right. Signl-intensities of single frtions were normlised to the highest vlue for eh protein. The frtions ontining the APC/C nd the MCC re indited (V 0 =void volume). Results representtive of two experiments. 5

6 α e α α rel. inding to the APC/C (time 0 =1) rel. inding to the APC/C (time 0 =1) mok retions sirna: relese (h) α APC α p omet α α α input GAPDH APC p omet tr IP APC3 IP GAPDH APC p omet M r uiquityltion retions * d APC/C f Cylin B1 -U(n) Cylin B1 (1-86) rel. inding to the APC/C (GAPDH = 1) si-p omet Autordiogrphy ound to the APC/C fter 45 min uiquityltion ontrol Figure S6 APC depletion prevents the relese of the MCC from the APC/C in vitro nd in vivo. () HeL ells were relesed from DMA-lok into fresh medium ontining 10 mm MG132. Smples were olleted t the indited time points nd the APC/C immunopreipitted using nti-apc3 ntiodies efore nlysis y immunolotting with the indited ntiodies. Results representtive of three experiments. Moleulr mss mrkers on the right. () Quntifitions of APC/C ound hekpoint proteins from the in vivo relese experiments shown in Fig. 5. The mounts of o-preipitted proteins were determined y quntittive immunolotting nd normlised to APC4 (men ± SEM from three experiments). () MCC-relese during in vitro uiquityltion retions using APC/C from prometphse ells immoilised on nti-apc3 eds. Mok retions ontin only uffer nd BSA. Uiquityltion retions ontin E1, UH10 nd UBE2s, ATP, uiquitin, ATP regenerting system nd His-tgged. After inution t o C for the indited time the retion mix ws removed, APC/C eds were wshed nd nlysed y immunolotting with the indited ntiodies. The sterisk (*) denotes His- tht inds to the APC/C during the retions. The lower nd is endogenous. Moleulr mss mrkers on the right. Results representtive of three experiments (d) Autordiogrphy of n in vitro uiquityltion ssy performed s in () ut with rdiolelled Cylin B1 (1-86) s sustrte. Note tht the whole retion mixture ws seprted y SDS-PAGE efore utordiogrphy. Moleulr mss mrkers on the right. Results representtive of three experiments. (e) Quntifitions of APC/C ound hekpoint proteins during in vitro uiquityltion retions shown in Fig. 5. The mount of o-preipitted proteins ws determined y quntittive immunolotting nd normlised to APC4. Grphs represent the men of two experiments. (f) Quntifitions of APC/C-ound hekpoint proteins fter 45 minutes inution in n in vitro uiquityltion retion. The mount of, nd ws determined s in (e) nd normlised to -treted retions (ontrol). Br hrts indite the men ± s.e.m. of three experiments. 6

0 100 50 NEBD to nphse n= 91 83 68 62 200 α APC3 α APC10 α pomet input (1/120) tr P APC3 IP APC4 IP α APC3 α APC α pomet input (1/400) tr IP APC3 IP 0 + + si-p omet + + si-p omet d α α α α APC input

7 NEBD to nphse n= α APC3 α APC10 α pomet input (1/120) tr P APC3 IP APC4 IP α APC3 α APC α pomet input (1/400) tr IP APC3 IP si-p omet + + si-p omet d α α α α APC input APC3 IP unound si-apc11 si-apc11 si-apc11 M r e APC/C Cylin B1 -U(n) Cylin B1 (1-86) mount of APC4: si-apc M r Autordiogrphy WB normlised ounts si-apc11 Cylin B1 uiquityltion Figure S7 Co-depleting APC- nd p omet delys nphse further even though APC depletion does not prevent p omet inding to the APC/C nd APC is not required for the tivity of mitoti APC/C in the sene of MCCs. () Time from NEBD to nphse of sirna-treted RPE1-Cylin B1-Venus ells. Stter dot lots show the men (red line) of the indited numer of ells from three experiments () Peptide-elutions of nti-apc3 nd nti-apc4 immunopreipittes from HeL ells rrested in prometphse were nlysed y immunolotting with the indited ntiodies. Moleulr mss mrkers on the right. Results representtive of three experiments. () Peptide-elutions of nti-apc3 immunopreipittes from HeL ells rrested in prometphse nd treted with the indited sirna oligonuleotides for 85h were nlysed y immunolotting with the indited ntiodies. Note tht more thn three mg extrt per immunopreipittion re required to visulise p omet inding to the APC/C. Moleulr mss mrkers on the right. Results representtive of three experiments. (d) HeL ells were relesed from doule thymidine-lok nd relese protool. After six hours 0.5 mm reversine ws dded followed y 10 mm MG132 one hour lter. Mitoti ells were hrvested y mitoti shke-off nd the APC/C immuno-preipitted with nti-apc3 ntiodies. The immunolot nlysis with the indited ntiodies shows the mounts of APC/C nd MCC tht re present in single in vitro uiquityltion retion for, nd si-apc11 retions. Note tht depleting APC does not inrese the mount of MCCs on the APC/C ompred to -tretment. Moleulr mss mrkers on the right. (e) In vitro uiquityltion ssys using the sme APC/C preipittes shown in (d) with s n tivtor nd Cylin B1 ( 1-86) s sustrte. For quntifition, the mount of uiquitylted Cylin B1 ws normlised to si- GAPDH retions nd orreted for the mount of APC4 present in individul retions s determined y quntittive immunolotting of the sme smples tht were used for the utordiogrphy (men ± s.e.m of four experiments). Moleulr mss mrkers on the right. 7

8 ΔK-GG ΔK-GG α α α / α α tuulin input tr IP si-apc3 si-apc6 d rel. inding to APC/C (t0 GAPDH = 1) APC4 IP unound si-apc3 si-apc6 si-apc3 si-apc6 Binding to the APC/C fter 30 min of relese si-uh10 () tuulin (50) rel. inding to APC/C (t0 GAPDH = 1) e rel. inding to APC/C (t0 = 1) 2.0 si-apc Binding to the APC/C fter 30 min of relese si-apc11 Relese from the APC/C si-uh10 Figure S8 is uiquitylted on lysine 490 in prometphse, APC3-, APC6- nd APC8-depletions do not inrese MCC-inding to the APC/C, nd APC11 ut not UH10-depleted ells re impired in the MCC relese from the APC/C. () CID frgment mss spetrum of SILAC-lelled peptide identifying uiquityltion site t K490 on APC4-ssoited CDC20 purified from prometphse ells. The uiquityltion site is determined y the mss differene of two glyine residues (DK-GG) on K490, the remnnt of trypti digestion of the CDC20-modified uiquitin hin. (see Supplementry Tle S2 online for individul mss hrge rtios (m/z). () Immunolot nlysis with the indited ntiodies of APC4 preipittes from the experiment shown in Fig 1d. Moleulr mss mrkers on the right. ( nd d). Quntifition of the mounts for MCCs on the APC4- or APC3- immunopreipittes shown in Fig. 7d nd Fig. 7e, respetively. The reltive mount of eh MCC-protein ws normlised to the initil mount of MCC on the APC/C from -treted ells efore the relese (t=0). Br hrts indite the men ± s.e.m. of three experiments nd three tehnil replites from Fig. 7d nd of three experiments from Fig. 7e. (e) Quntifition of the sme dt s in () ut normlised to the initil mount of MCCs present on the APC/C of the respetive sirna tretment. 8

9 MCC turnover during the SAC: APC/C MCC p omet MT Md1 pomet? U U U U X APC degrdtion U U U U APC po APC/C MCC turnover is prevented y depleting APC: pomet MT Md1 U U U U APC/C MCC Figure S9 APC nd p omet drive the turnover of the MCC during prometphse. () Untthed kinetohores generte wit-nphse signl y promoting the formtion of MCCs tht ind to the APC/C (APC/ C MCC ). MCC formtion is medited y Md1- dimeristion tht filittes the intertion of nd nd susequent reruitment of nd. APC nd p omet n relese nd from APC/C-ound nd -unound MCCs, respetively. During MCC turnover on the APC/C, dissoites nd is uiquitylted, relesed, nd susequently degrded y the 26S protesome. APC/C without MCC (poapc/c 28 ) is inhiited y new MCCs to omplete the yle. Protein X indites n unknown relese ftor or nother protein tht needs to e uiquitylted for the relese of MCCs, respetively. () Without APC, the MCC is loked onto the APC/C, unound MCCs umulte, nd relese nd degrdtion ut not uiquityltion is prevented. 9

Figure 1 α APC3 + α APC7 6 α APC 700 nm hnnel 800

APC HeL RPE1 Figure 3e M r α APC3 + APC6 6 Figure

(2nd detetion) 700 nm hnnel 800 nm hnnel 700 nm

APC α p omet α (2nd detetion) 700 nm hnnel 800 nm

Figure 6 M r α 200 α APC6 α APC11 α APC + α APC10

Figure 1 α APC3 6 α APC10 6 α APC 6 α APC 700 nm

(2nd detetion) α α α hnnel Figure 1d α APC6 α

(2nd detetion) α APC + α (2nd detetion) Figure 4

detetion) α p omet + α (2nd detetion) α p omet + α

Figure 7 M r Figure 7 α α Cylin A α 700 nm hnnel

APC10 r 6 α APC α APC11 700 nm hnnel 800 nm hnnel

hnnel si-p omet 700 nm hnnel 800 nm hnnel 700 nm

700 nm or 800 nm hnnels indite sns of the sme lot

re exited y 680 nm or 800 nm light, respetively.

10 Figure 1 α APC3 + α APC7 6 α APC 700 nm hnnel 800 nm hnnel Figure 2 M r α α tin α α tin 6 α APC α APC HeL RPE1 Figure 3e M r α APC3 + APC6 6 Figure 5 α Figure 6 M r α flg α Figure 7e M r α α α α APC (2nd detetion) 700 nm hnnel 800 nm hnnel 700 nm hnnel α 700 nm hnnel 800 nm hnnel (2nd detetion) α APC α p omet α (2nd detetion) 700 nm hnnel 800 nm hnnel 800 nm hnnel Figure 5 M r α (2nd detetion) Figure 6 M r α 200 α APC6 α APC11 α APC + α APC10 Figure 1 M r nmhnnel 800 nmhnnel α APC Figure 1 α APC3 6 α APC10 6 α APC 6 α APC 700 nm hnnel α + α p omet (2nd detetion) 700 nm hnnel (2nd detetion) α α α (2nd detetion) 700 nm hnnel 800 nm hnnel 700 nm hnnel Figure 1d α APC6 α APC3 +α α tuulin α APC8 (2nd detetion) 700 nm hnnel 800 nm hnnel 700 nm hnnel Figure 4 ( IP) α + (2nd detetion) α APC + α (2nd detetion) Figure 4 (APC4 IP) α + (2nd detetion) α APC + α (2nd detetion) α p omet + α (2nd detetion) α p omet + α (2nd detetion) Figure 4 M r α α α 800 nm hnnel Figure 7 M r Figure 7 α α Cylin A α 700 nm hnnel 800 nm hnnel α α (2nd detetion) 700 nm hnnel M α APC10 r 6 α APC α APC nm hnnel 800 nm hnnel Figure 7 α α α (2nd detetion) 700 nm hnnel 800 nm hnnel si-p omet 700 nm hnnel 800 nm hnnel 700 nm hnnel Figure S10 Complete sns of ll Western nlyses presented in Figs 1-7. Cropped regions re indited y dshed oxes. 700 nm or 800 nm hnnels indite sns of the sme lot using seondry ntiodies oupled to flourophores tht re exited y 680 nm or 800 nm light, respetively. 10

11 Supplementry Tles Tle S1 Sttistil nlyses. Dt derived from time-lpse mirosopy nd immunopreipittion experiments presented in the indited figures were nlysed y unpired Mnn-Whitney or pired Student s t tests, respetively. The sterisk (*) indites dt sets where signifine ws not determined s the dt inluded ells tht did not initite nphse during the oservtion. Tle S2 CID frgment ion tle of the SILAC-lelled peptide identifying uiquityltion site t K490. Mss/hrge rtios of + (red) nd y+ ions (lue) s indited on the CID mss spetrum shown in Supplementry Figure S

dsrna GFP 0 Ca 0 Ca 0 Ca TG Iono Time (s)

dsrna GFP 0 Ca 0 Ca 0 Ca TG Iono Time (s) Rtio (FL1/FL3) MFI dsrna GFP C dsrna dori C 1 2 1 2 Rtio (FL1/FL3) MFI C 1 2 Rtio (FL1/FL3) MFI C 1 2 C 1 2 C 1 2 Supplementry Figure 1. RNAi-medited depletion of dori hs no effet on the filling stte of