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5. MATERIAL  AND  METHODS

5.7. Xenopus  oocyte  injections

  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.  

5.7. Xenopus  oocyte  injections  

Oocytes  were  obtained,  cultured  and  injected  as  described  previously  (Ohe  et   al.,   2007).   Briefly,  Xenopus   stage   VI   oocytes   were   injected   with   10nL   of   an   8mg/ml   UbcX   protein   solution   or   buffer   and   maturation   was   induced   with   progesterone  (5  μg/ml;  SIGMA).  Oocytes  undergoing  GVBD  in  a  time  window   of  15  min  were  pooled  and  at  the  indicated  time  points  after  GVBD  collected   and   snap   frozen   in   liquid   N2.   Oocytes   were   homogenized   in   CIP-­‐buffer   containing   1x   complete   protease   inhibitors   (Roche)   and   incubated   with   10   units   CIP   (NEB)   for   30’   at   20°C.   An   equal   volume   of   3x   laemmli   buffer   was   added  and  the  samples  were  analyzed  by  WB.  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                              Literature  

 

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6. LITERATURE  

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Alberts,   B.,   Johnson,   A.,   Lewis,   J.,   Raff,   M.,   and   Roberts,   K.a.W.,   P.   (2002).  

Molecular  Biology  of  the  Cell,  Vol  4th  edition  (New  York,  Garland  Sciences).  

Allers,   T.,   and   Lichten,   M.   (2001).   Differential   timing   and   control   of   noncrossover  and  crossover  recombination  during  meiosis.  Cell  106,  47-­‐57.  

Berlingieri,  M.T.,  Pallante,  P.,  Sboner,  A.,  Barbareschi,  M.,  Bianco,  M.,  Ferraro,   A.,   Mansueto,   G.,   Borbone,   E.,   Guerriero,   E.,   Troncone,   G.,   et   al.   (2007).  

UbcH10   is   overexpressed   in   malignant   breast   carcinomas.   Eur   J   Cancer   43,   2729-­‐2735.  

Bhatt,   R.R.,   and   Ferrell,   J.E.,   Jr.   (1999).   The   protein   kinase   p90   rsk   as   an   essential  mediator  of  cytostatic  factor  activity.  Science  286,  1362-­‐1365.  

Buonomo,   S.B.,   Clyne,   R.K.,   Fuchs,   J.,   Loidl,   J.,   Uhlmann,   F.,   and   Nasmyth,   K.  

(2000).   Disjunction   of   homologous   chromosomes   in   meiosis   I   depends   on   proteolytic  cleavage  of  the  meiotic  cohesin  Rec8  by  separin.  Cell  103,  387-­‐398.  

Burnette,  W.N.  (1981).  "Western  blotting":  electrophoretic  transfer  of  proteins   from  sodium  dodecyl  sulfate-­‐-­‐polyacrylamide  gels  to  unmodified  nitrocellulose   and   radiographic   detection   with   antibody   and   radioiodinated   protein   A.   Anal   Biochem  112,  195-­‐203.  

Chen,   R.H.,   Shevchenko,   A.,   Mann,   M.,   and   Murray,   A.W.   (1998).   Spindle   checkpoint   protein   Xmad1   recruits   Xmad2   to   unattached   kinetochores.   J   Cell   Biol  143,  283-­‐295.  

Chen,  R.H.,  Waters,  J.C.,  Salmon,  E.D.,  and  Murray,  A.W.  (1996).  Association  of   spindle   assembly   checkpoint   component   XMAD2   with   unattached   kinetochores.  Science  274,  242-­‐246.  

 

 

 

 

promoting  complex/cyclosome  as  a  pseudosubstrate  inhibitor.  Genes  Dev  20,   2410-­‐2420.  

Minshull,   J.,   Sun,   H.,   Tonks,   N.K.,   and   Murray,   A.W.   (1994).   A   MAP   kinase-­‐

dependent  spindle  assembly  checkpoint  in  Xenopus  egg  extracts.  Cell  79,  475-­‐

486.  

  complex/cyclosome  absolutely  requires  Emi2  binding  via  the  C-­‐terminal  RL  tail.  

Mol  Biol  Cell  21,  905-­‐913.  

Okamoto,   Y.,   Ozaki,   T.,   Miyazaki,   K.,   Aoyama,   M.,   Miyazaki,   M.,   and   Nakagawara,  A.  (2003).  UbcH10  is  the  cancer-­‐related  E2  ubiquitin-­‐conjugating   enzyme.  Cancer  Res  63,  4167-­‐4173.  

 

Ubiquitination  by  the  anaphase-­‐promoting  complex  drives  spindle  checkpoint   inactivation.  Nature  446,  921-­‐925.  

  anaphase-­‐promoting   complex/cyclosome   through   a   D-­‐box-­‐independent   mechanism.  Mol  Biol  Cell  21,  2589-­‐2597.  

 

(1997).   Dominant-­‐negative   cyclin-­‐selective   ubiquitin   carrier   protein   E2-­‐

C/UbcH10  blocks  cells  in  metaphase.  Proc  Natl  Acad  Sci  U  S  A  94,  2362-­‐2367.  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                              Literature  

  mediated   regulation   of   Emi2   in   the   maintenance   of   CSF   arrest.   Curr   Biol   17,   213-­‐224.  

Wu,   T.,   Merbl,   Y.,   Huo,   Y.,   Gallop,   J.L.,   Tzur,   A.,   and   Kirschner,   M.W.   (2010).  

UBE2S   drives   elongation   of   K11-­‐linked   ubiquitin   chains   by   the   anaphase-­‐

promoting  complex.  Proc  Natl  Acad  Sci  U  S  A  107,  1355-­‐1360.  

Yu,  H.,  King,  R.W.,  Peters,  J.M.,  and  Kirschner,  M.W.  (1996).  Identification  of  a   novel  ubiquitin-­‐conjugating  enzyme  involved  in  mitotic  cyclin  degradation.  Curr   Biol  6,  455-­‐466.  

Zachariae,  W.,  and  Nasmyth,  K.  (1999).  Whose  end  is  destruction:  cell  division   and  the  anaphase-­‐promoting  complex.  Genes  Dev  13,  2039-­‐2058.  

Zou,  H.,  McGarry,  T.J.,  Bernal,  T.,  and  Kirschner,  M.W.  (1999).  Identification  of   a   vertebrate   sister-­‐chromatid   separation   inhibitor   involved   in   transformation   and  tumorigenesis.  Science  285,  418-­‐422.  

 

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                              Appendix  

 

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7. APPENDIX  

7.1. Summary  

Mature  Xenopus  oocytes  are  arrested  in  meiosis  by  the  activity  of  XErp1/Emi2,   an   inhibitor   of   the   ubiquitin-­‐ligase   anaphase-­‐promoting   complex/cyclosome   (APC/C).  Upon  fertilization,  XErp1  is  degraded  resulting  in  APC/C  activation  and   the  consequent  degradation  of  cell  cycle  regulators  and  exit  from  meiosis.  In   this   study,   we   show   that   a   modest   increase   in   the   activity   of   the   ubiquitin-­‐

conjugating  enzyme  UbcX  overrides  the  meiotic  arrest  in  an  APC/C-­‐dependent   reaction.   Intriguingly,   XErp1   remains   stable   under   these   conditions.   We   observed   that   UbcX   causes   the   ubiquitylation   of   XErp1   followed   by   its   dissociation   from   the   APC/C.   Our   data   favor   the   idea   that   ubiquitylation   regulates   XErp1’s   APC/C   inhibitory   activity.   Similarly,   UbcX   can   trigger   activation   of   the   APC/C   in  Xenopus   egg   extracts   with   an   activated   spindle   checkpoint.  This  suggests  that  in  vertebrate  cells,  the  APC/C  can  liberate  itself   from   XErp1   and   spindle   checkpoint   inhibition   in   an   autocatalytic   manner.   In   addition,   our   findings   that   Cdc20   is   stable   during   meiotic   arrest   suggest   that   the  meiotic  arrest  is  not  mediated  via  the  degradation  of  Cdc20.    

7.2. Zusammenfassung  

In   Xenopus   laevis   werden   reife   Eizellen   durch   die   Aktivität   des   Anaphase   promoting   complex/cyclosome   (APC/C)   Inhibitors   XErp1   in   der   Meiose   arretiert.   Die   Befruchtung   des   Eies   führt   zum   Abbau   von   XErp1   und   dadurch   zur  Aktivierung  des  APC/Cs.  Der  APC/C  baut  nun  Regulatoren  des  Zellzyklus  ab   und  die  Eizellen  können  die  Meiose  verlassen.  Wir  zeigen  in  dieser  Studie,  dass   eine  geringe  Erhöhung  der  UbcX  Aktivität  ausreicht  um  den  meiotischen  Arrest   in  einer  APC/C  abhängigen  Reaktion  zu  verlassen.  Interessanterweise  bewirkt   die  Zugabe  von  UbcX  nicht  den  Abbau,  sondern  die  Ubiquitylierung  von  XErp1,  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                              Appendix  

  welche   von   der   Dissoziation   von   XErp1   vom   APC/C   begleitet   wird.   Unsere   Daten   deuten   darauf   hin,   dass   die   APC/C   inhibierende   Aktivität   von   XErp1   durch  Ubiquitylierung  reguliert  werden  kann.  In  einer  ähnlichen  Art  und  Weise   kann   UbcX   in  Xenopus   Eiextrakten   den   Spindelkontrollpunkt   übergehen   und   den  APC/C  aktivieren.    Daraus  lässt  sich  die  These  ableiten,  dass  sich  der  APC/C   durch   seine   autokatalytische   Aktivität   von   der   Inhibierung   durch   XErp1   oder   den   Spindelkontrollpunkt   befreien   kann.   Weiters   zeigen   unsere   Ergebnisse,   dass   Cdc20   während   des   meiotischen   Arrests   stabil   ist.   Daher   ist   es   naheliegend,   dass   der   meiotische   Arrest   nicht   durch   den   Abbau   von   Cdc20   herbeigeführt  wird.    

7.3. Acknowledgements  

In  this  part  of  the  thesis  I  would  like  to  tell  you  how  grateful  I  am  to  the  many   extraordinary  people  I  had  the  opportunity  to  work  with  along  the  way  of  my   doctoral  studies.  

First,   I   wish   to   thank   my   advisor,   Thomas   U.   Mayer   for   all   his   guidance,   encouragement,  support,  and  patience.  His  sincere  interests  in  science  and  his   way  of  approaching  scientific  questions  have  been  a  great  inspiration  to  me.

   

Also,  I  would  like  to  thank  Prof.  Elke  Deuerling,  Prof.  Martin  Scheffner  and  Prof.  

Olaf  Stemmann  for  reviewing  my  thesis.    

Additionally,  I  would  like  to  acknowledge  all  the  present  and  past  TUMs-­‐  my   friends   and   colleagues,   for   their   very   helpful   insights,   comments   and   suggestions  as  well  as  for  providing  technical  support  and  assistance.  Thomas   Tischer  my  smart  and  patient  co-­‐author;  Tobias  and  Andi  for  teaching  me  and   helping   me   to   design   my   first   killer   experiments;   Hümmi   for   setting   high   working  standards  and  helping  me  to  do  my  best  to  fulfill  them;  Die  Jenny,  my   friend  for  the  fun  in  the  lab;  Johanna,  for  being  tough,  cool  and  fun  at  the  same   time;   Julia   for   taking   care   of   the   little   things   in   the   lab;   Lucia   for   emergency   support;  Marianne,  our  artist,  for  all  the  help  with  various  paperwork;  Martina  

    UbcX  dissociates  XErp1  from  the  APC/C                                                                                                                                                              Appendix  

 

77   for  teaching  me  and  showing  me  so  many  new  and  old-­‐school  things,  Mario  for   always  being  there  for  me  and  supporting  me,  die  Moni,  for  being  Moni-­‐  sweet   and   a   bit   chaotic   and   for   keeping   the   secrets;   Stefan   for   his   great   sense   of   humor   and   computer   advice;   Violeta   for   keeping   me   up   to   date   with   the   literature   and   providing   french   sweets;   Wiebke   for   the   nice,   but   short   time;  

Our  smart  diploma  students  Susanna,  Elena  and  Judith;  The  kids:  Annalein,  for   her  help  and  fun;  Martin  “Martinpedia”  for  cool  discussions,  Norris  for  helping   me  to  improve  my  patience  and  Saurav  for  letting  me  be  his  teacher  and  for   the   loads   of   fun;   my   VTK   students   Susanna,   Falk,   Anja,   Ulrike,   Andreas   and   Alex,  as  well  as  the  members  of  the  Deuerling  lab,  especially  Miriam,  Steffen,   Annika,  Renate,  Marcel  and  Rainer.  

I  am  very  thankful  my  friends,  especially  Mirijam,  Jenny  and  Gerrit  as  well  as   Anna,  Eunice,  Tom  and  Louise,  Sabine  and  Mikael.    

A  particular  acknowledgement  to  my  partner  Mario,  because  he  taught  me  to   never   stop   laughing   and   that   any   difficulties   can   be   overcome   when   we   approach  them  one  step  at  a  time.    

I   must   also   thank   my   loving   mother   and   my   father,   brother   and   sisters,   my   nephew  and  nieces,  aunts,  uncles  and  cousins  who  have  given  me  their  fullest   support.    

Finally,  I  dedicate  this  work  to  my  uncle,  Karl-­‐Heinz  Krischke,  who  believes  in   diligence,   science   and   the   pursuit   of   academic   excellence,   and   most   importantly,  in  me.    

 

Title  of  the  thesis:  “Non-­‐proteolytic  ubiquitylation  regulates  the   APC/C-­‐inhibitory  function  of  XErp1.”  

proteolytic  ubiquitylation  counteracts  the  APC/C-­‐inhibitory  function  of  XErp1.  

EMBO   Rep.   2011   Mar   11.   [Epub   ahead   of   print],   PMID:   21399619;   *equal   contribution  

Tischer   T*.,  Hörmanseder   E*.     and   Mayer   T.U.   The   APC/C   inhibitor   XErp1   is   essential   for   early   embryonic   divisions   (Science,   under   revisions).   *equal   contribution