SP Mechanisms of Genome Haploidization -Regulatory Mechanisms of Meiosis in Vertebrate Oocytes-
Meiosis is the process by which haploid germ cells – also called gametes – are formed from diploid
somatic precursor cells. To create haploid gametes, meiosis proceeds through two consecutive rounds of nuclear divisions, meiosis I and meiosis II, without an intervening S-phase. In most vertebrates, mature eggs await fertilization arrested at metaphase of meiosis II. Recently, we identified XErp1 (Xenopus Emi1 related protein) as the main factor mediating metaphase II arrest. XErp1 prevents anaphase onset by inhibiting the ubiquitin-ligase APC/C (anaphase promoting complex/cyclosome) whose activity is required to target cell cycle regulators for destruction. Interestingly, XErp1’s inhibitory function on the APC/C depends on its zinc-binding region but not its F-box. Typically, F-box proteins are components of SCF (Skp1/Cul/F-box protein) ubiquitin ligase complexes and are responsible for targeting phosphorylated substrates for proteasome-dependent destruction.
To gain insights into the regulation of meiotic maturation, we would like to address the following questions:
i) What is the function of XErp1 as an F-box protein?
ii) What are the general functions of SCFs in
iii) How does ubiquitylation/deubiquitylation of CDC20 affect meiotic progression?
To address these questions, we will first perform micro-injection experiments to analyze meiotic maturation of Xenopus oocytes expressing F-box mutant proteins, starting with the F-box mutant of XErp1. Microscopic and biochemical studies will determine which meiotic process is affected by the inactivation of specific Fbox proteins. The identification of interesting F-box proteins will then be followed by the investigation of their substrates critical for meiotic progression. To this end, we will apply an in vitro expression cloning (IVEC) approach as well as quantitative mass spectroscopy analyses. In vitro SCF-ubiquitylation assays will confirm the identified substrates. The identification of relevant kinases mediating the recognition of substrates by SCFs will provide important insights into the temporal regulation of SCF-mediated protein degradation in meiosis and, thus, into the regulatory network controlling entry into and progression through meiosis. Additionally, we will investigate the function of ubiquitylation/deubiquitylation of CDC20, the only known co-activator of the APC/C in female Xenopus meiosis. Our preliminary work revealed that ectopic UbcH10, which reportedly mediates the ubiquitylation of CDC20, triggers release from metaphase II. Further studies will investigate how this regulatory mechanism controls APC/C activity during meiotic progression. Furthermore, we will apply this mechanism to gain insights into XErp1’s inhibitory mode of
action on the APC/C which so far remains elusive.
- FB Biologie
|(2011): Non-proteolytic ubiquitylation counteracts the APC/C-inhibitory function of XErp1 EMBO reports ; 12 (2011), 5. - S. 436-443. - ISSN 1469-221X. - eISSN 1469-3178||
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). On 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 in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1.
|Laufzeit:||01.12.2009 – 31.05.2012|