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5. MATERIAL  AND  METHODS

5.6. Xenopus  egg  extracts

  UbcX  dissociates  XErp1  from  the  APC/C                                                                                                        Material  and  Methods  

 

61   was  performed  using  specific  primary  antibodies  (see  Table  4.3),  commercially   available  HRP-­‐conjugated  secondary  antibodies  (horse  radish  peroxidase;  Bio-­‐

Rad  and  Dianova)  and  ECL  (100  mM  Tris-­‐HCl  pH  8.5,  1.25  mM  luminol,  225  μM   coumaric  acid,  0.015%  v/v  H2O2)  or  ECL  plus  (Lumigen  TMA-­‐6,  GE  Healthcare)   reagents.  

5.6. Xenopus  egg  extracts  

5.6.1. Xenopus  CSF  egg  extract  preparation    

Three  days  before  extract  preparation,  female  Xenopus  laevis  were  primed  by   injecting  50  U  PMSG  (gonadotropin,  pregnant  mare  serum  in  H2O,  Merck)  into   the  dorsal  lymph  sack.  One  day  before  extract  preparation,  frogs  were  induced   to   ovulate   by   the   injection   of   800   U   HCG   (human   chorionic   gonadotropin   in   H2O,  Sigma)  into  the  dorsal  lymph  sack.  Frogs  were  put  into  tanks  filled  with  2L   of  1x  MMR  [0.1  M  NaCl,  2  mM  KCl,  1  mM  MgSO4,  2  mM  CaCl2,  0.1  mM  EDTA,  5   mM  HEPES  pH  7.8]  for  ovulation.  Laid  eggs  of  sufficient  quality  were  collected,   washed  with  2L  1x  MMR  and  treated  with  dejellying  solution  for  up  to  seven   minutes.  Eggs  were  then  washed  with  1L  CSF-­‐XB  [2  mM  MgCl2,  0.1  mM  CaCl2,   100  mM  KCl,  5  mM  EGTA,  50  mM  sucrose,  10  mM  HEPES  pH  7.7].  Activated   and   lysed   eggs   were   removed   and   the   eggs   were   transferred   into   centrifugation   tubes   pre-­‐filled   with   1   ml   CSF-­‐XB   containing   0,1   mg/ml   cytochalasin  B  (Sigma).  The  eggs  were  compacted  by  a  two-­‐step  centrifugation   for  1  minute  at  160xg  and  another  minute  at  650xg  in  a  swing  out  rotor.  Excess   buffer  was  removed  from  the  tube  after  centrifugation  leaving  the  compacted,   eggs   behind.   In   a   final   centrifugation   step   the   eggs   were   then   lysed   by   centrifugation  for  10  minutes  at  17.000xg  at  4  °C.  The  cytoplasmic  layer  was   then  isolated  with  a  1  ml  syringe  by  puncturing  the  tube  at  the  bottom  of  this   layer.   The   extract   was   supplemented   cytochalasin   B   (10µg/ml,   Sigma)   and   stored  on  ice  for  further  use.  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                        Material  and  Methods  

  5.6.2. Extract  manipulations  

All   incubations   were   done   at   20°C.   When   indicated,   CaCl2   was   added   to   600   µM,  cycloheximide  to  350  µM,  MG262  to  100  µM,  nocodazole  to  33  µM  and   Ocadaic   acid   to   2   µM.   “High   sperm”   and   “low   sperm”   extract   contained   11,5x106  and  1x106,  respectively,  sperm  nuclei  per  ml  extract.  In  general,  6xhis-­‐

UbcH10wt,  6xhis-­‐UbcH10ci  were  added  to  a  final  concentration  of  12  µM,  6xhis-­‐

UbcXwt  or  6xhis-­‐UbcXci  to  6  µM  (unless  explicitly  mentioned),  MBP-­‐XErp1  CT  (aa   374-­‐651)  to  200nM,  Ubiquitin-­‐aldehyde  (Boston  Biochemicals)  to  6  µM  and  a   corresponding   volume   of   dialysis   buffer   was   used   as   buffer   control.   To   dephosphorylate  extract  proteins,  samples  were  diluted  with  9  volumes  of  1x   CIP-­‐buffer   (NEB)   containing   10   units   CIP   (NEB)   and   incubated   for   30   min   at   25°C.   For   glycerol-­‐gradient   centrifugation,   extracts   were   cleared   by   centrifugation   at   186.000xg   and   resolved   on   10–40%   glycerol   gradient   by   centrifugation   at   150.000xg.   0.5   ml   fractions   were   collected   and   analyzed   by   SDS-­‐PAGE  and  immunoblotting.  

5.6.3. Immunodepletion    

For  the  depletion  of  100µl  egg  extract,  3x10  µg  Cdc20,  Cdc27  or  approximately   3x15  µg  βTRCP  antibodies  as  well  as  the  corresponding  control  antibodies  were   coupled   to   3   x   25   µl   protein   A   Dynabeads   (Pierce)   in   100µl   PBS-­‐Tx   [1xPBS,   0,1%TritonX100]   for   1h   at   room   temperature   rotating   on   a   wheel.   The   antibody   beads   were   washed   with   500µl   PBS-­‐Tx,   500µl   PBS-­‐Tx   containing   500mM  NaCl  and  3x  with  500µl  CSF-­‐XB  [2  mM  MgCl2,  0.1  mM  CaCl2,  100  mM   KCl,  5  mM  EGTA,  50  mM  sucrose,  10  mM  HEPES  pH  7.7].  Every  time,  the  beads   were  collected  by  putting  them  in  the  testube  on  a  magnet;  only  after  the  last   washing  step,  the  beads  were  additionally  centrifuged  for  10  s  in  a  nanofuge   and  all  residual  buffer  was  removed  carefully.  Next,  the  extract  was  incubated   with   the   antibody   beads   in   three   rounds   for   30   min   on   ice.   Every   time   the   beads  were  removed  from  the  extract  by  incubating  the  test  tube  for  10  min  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                        Material  and  Methods  

 

63   on  ice  on  the  magnet,  and  the  extract  was  added  to  new  antibody-­‐beads.  After   the   third   round,   the   beads   were   carefully   removed   and   the   depleted   extract   was  used  for  further  experiments.    

5.6.4. Immunoprecipitation    

For   Cdc27   immunoprecipitation   assays,   the   250   µl   extracts   were   treated   as   indicated,  diluted  1:1  with  CSF-­‐XB  buffer  and  incubated  with  5  µg  anti-­‐Cdc27   antibodies   coupled   to   30   µl   protein   A   agarose   beads.   The   reaction   was   incubated   for   4   h   at   4°C   rotating   on   a   wheel.   The   beads   were   retrieved   by   centrifugation,   washed   three   times   with   1   ml   CSF-­‐XB   and   resuspended   in   3x   laemmli   buffer   for   westernblot   analysis.   To   quantify   immunoprecipitated   Cdc27   and   XErp1,   immunoblots   were   visualized   with   ECL   reagent.   Signal   intensities   were   quantified   using   Image   J   software.   Anti-­‐Cdc27   immunoprecipitates  were  used  as  loading  controls  and  the  relative  amounts  of   copurified  XErp1  were  calculated.    

XErp1   and   Cdc20   immunoprecipitation   assays   were   performed   like   described   for  Cdc27  immunoprecipitations,  except  125  µM  N-­‐ethylmaleimide  was  added   when  the  extracts  were  diluted  with  CSF-­‐XB.    

For  the  purification  of  his-­‐tagged  ubiquitin  conjugates  a  protocol  described  in   (Glockzin  et  al.,  2003)  was  modified  with  the  help  of  Alejandro  Rojas.  Briefly,  to   CSF   extract   12,5   µM   6xhis-­‐Ubiquitin   or   6xhis-­‐UbiquitinDGG   was   added   and   incubated  with  6xhis-­‐UbcXwt,  6xhis-­‐UbcXci  or  buffer  at  20°C  for  30’.  The  extract   was   denatured   at   4°C   by   adding   9   volumes   of   denaturation   buffer   (6M   guanidinium-­‐HCl,  100  mM  Na2HPO4/NaH2PO4  pH  8.0,  10  mM  imidazole,  10  mM   β-­‐mercaptoethanol   and   complete   protease   inhibitors   (Roche)),   cleared   by   centrifugation  and  incubated  with  Ni2+-­‐NTA  beads  (Quiagen).  The  beads  were   washed,   the   bound   proteins   were   re-­‐natured,   eluted   into   3x   laemmli   buffer   containing  200mM  Imidazole  and  analyzed  by  Western  blotting.  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                        Material  and  Methods  

  5.6.5. In  vitro  ubiquitylation  assays  

APC/C   was   immunoprecipitated   from   2   ml   CSF   extract   using   25   µg   Cdc27   antibodies  immobilized  on  protein  G  dynabeads  (Invitrogen).  Assays  were  done   as  described  previously  (Schmidt  et  al.,  2005),  except  that  no  exogenous  Cdc20   or  XErp1  was  added:  The  beads  were  washed  once  in  QA  [100  mM  KCl,  1  mM   MgCl2,  1  mM  CaCl2,  10  mM  Tris  pH  7,5]  and  twice  in  CSF-­‐XB  and  divided  into   10.  A  mix  containing  E1  (110  nM;  Boston  Biochemicals),  Ubiquitin  (1,25  mg/ml;  

Boston  Biochemicals)  and  an  energy  regeneration  system  [1  mM  ATP,  20  mM   creatinephosphate,   150   µg   /ml   creatine-­‐phospho-­‐kinase   (Sigma)]   and   UbcX   (12,5   µM)   in   CSF-­‐XB   buffer   was   added   and   the   reaction   was   started   by   the   addition   of   UbcX.   For   Cdc20   ubiquitylation   assays,   an   equal   volume   of   3x   laemmli  buffer  was  added  to  the  reaction  at  the  indicated  time  points.  To  assay   XErp1   ubiquitylation   and   release   from   the   APC/C,   the   beads   were   separated   from  the  supernatant  on  a  magnet  and  the  supernatant  was  added  to  an  equal   amount  of  3x  laemmli  buffer.  Samples  were  directly  processed  and  analyzed  by   WB.