Abstract
In the fission yeast, Schizosaccharomyces pombe, synaptonemal complexes (SCs) are not formed during meiotic prophase. However, structures resembling the axial elements of SCs, the so-called linear elements (LinEs) appear. By in situ immunostaining, we found Pmt3 (S. pombe's SUMO protein) transiently along LinEs, suggesting that SUMOylation of some component(s) of LinEs occurs during meiosis. Mutation of the SUMO ligase Pli1 caused aberrant LinE formation and reduced genetic recombination indicating a role for SUMOylation of LinEs for the regulation of meiotic recombination. Western blot analysis of TAP-tagged Rec10 demonstrated that there is a Pli1-dependent posttranslational modification of this protein, which is a major LinE component and a distant homolog of the SC protein Red1. Mass spectrometry (MS) analysis revealed that Rec10 is both phosphorylated and ubiquitylated, but no evidence for SUMOylation of Rec10 was found. These findings indicate that the regulation of LinE and Rec10 function is modulated by Pli1-dependent SUMOylation of LinE protein(s) which directly or indirectly regulates Rec10 modification. On the side, MS analysis confirmed the interaction of Rec10 with the known LinE components Rec25, Rec27, and Hop1 and identified the meiotically upregulated protein Mug20 as a novel putative LinE-associated protein.
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Acknowledgments
The meiotic cDNA library was kindly provided by Hiroshi Nojima (Osaka University) and strains and plasmids by Kathy Gould (Vanderbilt University, Nashville), Alexander Lorenz (University of Oxford), Cristina Martín-Castellanos (Universidad de Salamanca), Gerald Smith (Fred Hutchinson Cancer Research Center, Seattle), Takashi Toda (Cancer Research UK, London), and Matthew Whitby (University of Oxford). We gratefully acknowledge the help of Lubos Cipak and the Ammerer lab (MFPL Vienna) with biochemical experiments and Maria Novatchkova (IMP Vienna) with bioinformatics, and we are grateful to Hubert Renauld (Vienna Medical University) for valuable suggestions. This work was supported by grant no. P18186 from the Austrian Science Fund (FWF) and by grant no. I031-B from the University of Vienna.
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Supplemental Table S1
Strain list (DOC 61 kb)
Supplemental Fig. S2
Treatment with calf intestinal phosphatase (CIP) does not notably reduce the amount of high-molecular-weight Rec10 species, suggesting that the slower migrating forms of Rec10 are not (only) due to phosphorylation. As a positive control for CIP activity, the size reduction of S. cerevisiae Mks1 protein in the presence of CIP is shown. TCA protein extracts were prepared as described for whole-protein isolation (in the “Materials and methods” section) except that the TCA pellets were neutralized by washing with 2M Tris pH8.0 and resuspended in 10 mM Tris buffer pH8.0. The whole proteins were incubated with or without CIP (Roche) for 1 h at 37°C in the corresponding buffer supplied by manufacturer. The procedure was described by Sekito et al. (2002, Mol. Biol. Cell 13:795-804). S. cerevisiae Mks1 is known to be phosphorylated (Sekito et al. 2002; Ferreira Júnior et al. 2005; Gene 354:2-8PSY142). Cells were grown in YPD medium and 3× HA- and 12× His-tagged Mks1 was detected on the Western blot with anti-HA antiserum (DOC 134 kb)
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Spirek, M., Estreicher, A., Csaszar, E. et al. SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe . Chromosoma 119, 59–72 (2010). https://doi.org/10.1007/s00412-009-0241-5
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DOI: https://doi.org/10.1007/s00412-009-0241-5