Expression and localization of Rdd proteins in<i>Xenopus</i>embryo

Lim, Jong-Chan; Kurihara, Sayaka; Tamaki, Rie; Mashima, Yutaka; Maéno, Mitsugu

doi:10.5115/acb.2014.47.1.18

Cited by 3 publications

(3 citation statements)

References 38 publications

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“…We surmise that these are released as well, rather than degraded (Dataset S1, Table S3). One example is Riddle4 (60) with known functions reported later in development (61). While the secretion of proteases is a well-recognized mechanism, the release of endogenous protease inhibitors in response to fertilization was not known.…”

Section: Significancementioning

confidence: 99%

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Presler

Itallie

Klein

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

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Section: Significancementioning

confidence: 99%

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Presler

Itallie

Klein

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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show abstract

“…We surmise that these are released as well, rather than degraded (Table S3). One example is Riddle4 (60) with known functions reported later in development (61). While the secretion of proteases is a well-recognized mechanism, the release of endogenous protease inhibitors in response to fertilization was not known.…”

Section: Proteins Released From the Eggmentioning

confidence: 99%

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in theXenopusegg

Presler

Itallie

Klein

et al. 2017

Preprint

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SummaryFertilization triggers release from meiotic arrest and initiates events that prepare for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site phosphorylation. Overall, the data suggest that degradation is limited to a few low abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium activated kinases. We identify putative degradation targets as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

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“…For detection of GFP expression in the reporter DNA-injected oocytes, western blot analysis was performed as described previously (Lim et al, 2014). At 24 h after DNA injection, oocytes were added to a cell lysis buffer (0.1 M KCl, 2 mM MgCl 2 , 1 mM phenylmethanesulfonyl fluoride (PMSF), 10 mM HEPES − KOH, pH 7.4) and were homogenized with a disposable pestle (Bio-Masher, Sarstedt Co. Ltd, Tokyo, Japan).…”

Section: Western Blot Analysismentioning

confidence: 99%

Specific activation of thehb4gene in theXenopusoocyte through a Nobox-binding element located at the proximal promoter sequence

Nakamigawa

Kondo

Maéno

2019

Zygote

Self Cite

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SummaryWe isolated and characterized Xenopus tropicalis hb4 flanking DNA and showed that the −3076/+29 sequence was able to drive stage-specific transcription in the developmental process. Transgenic reporter analysis indicated that green fluorescent protein was expressed in the ovaries of female frogs at 3 months of age and in both the ovaries and testis of frogs at 6 months of age. A series of experiments with deletion of the flanking sequence and a subsequent luciferase reporter assay revealed that there were two positive regulatory regions and that the most proximal sequence of the promoter region had a certain level of transcriptional activity in oocytes. Subsequently, we showed that a conserved sequence containing Nobox-binding element (NBE) was essential for transcriptional activation and that Nobox expressed in the ovary had a crucial role in hb4 transcription through the NBE sequence.

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Expression and localization of Rdd proteins inXenopusembryo

Cited by 3 publications

References 38 publications

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in theXenopusegg

Specific activation of thehb4gene in theXenopusoocyte through a Nobox-binding element located at the proximal promoter sequence

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