Localization of the Drosophila Rad9 protein to the nuclear membrane is regulated by the C-terminal region and is affected in the meiotic checkpoint.
Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint.
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Rotem
R
Kadir
Anna
A
Bakhrat
Ronit
R
Tokarsky
Uri
U
Abdu
D000818:Animals; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology* / Q000378:metabolism; D005260:Female; D008540:Meiosis; D009685:Nuclear Envelope / Q000378:metabolism*; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism; D009866:Oogenesis; D006080:Ovarian Follicle / Q000166:cytology; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
C501957:BAF protein, Drosophila; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D012333:RNA, Messenger; C069930:rad9 protein