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.
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6. LITERATURE
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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