Yeast two-hybrid system identifies the ubiquitin-conjugating enzyme mUbc9 as a potential partner of mouse Dac
Yeast two-hybrid system identifies the ubiquitin-conjugating enzyme mUbc9 as a potential partner of mouse Dac
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DOI：
10.1016/s0925-4773(00)00402-0
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发表时间：
2000-10-01
影响因子：
2.6
通讯作者：
Krauss, S
中科院分区：
文献类型：
--
作者：
Machon, O;Backman, M;Krauss, S
使用酵母双杂交系统和下拉实验，我们证明，小鼠Dac（mDac）特异性结合小鼠泛素结合酶mUbc 9。与果蝇腊肠犬（dac）和无眼（eya）基因产物之间的直接相互作用相反，我们无法通过相同的方法检测到mDac与mEya 2（果蝇Eya的功能性小鼠同源物）结合。用mDac转染的各种细胞系的免疫染色显示，mDac蛋白主要存在于细胞核中，但易位至细胞质并以细胞周期依赖性方式沿着核膜浓缩。mDac的缺失分析显示细胞内定位和蛋白稳定性与mUbc 9的结合相关。与mUbc 9相关的mDac的C-末端一半保持在细胞质中并在蛋白酶体中降解，而非相互作用的N-末端仅在细胞核中，并且比全长mDac或其C-末端部分更稳定。在整装胚胎或组织切片上的原位杂交检测与mDac表达互补和重叠区域中的mUbc 9转录物，特别是在肢芽、脊髓和前脑的增殖区中。用抗mDac抗体染色的小鼠胚胎证明mDac定位于细胞核和细胞质中，在迁移的神经嵴细胞中以细胞质为主。在增殖区中，未形成可见的核膜，并且在整个细胞中检测到mDac。(C)2000爱思唯尔科学爱尔兰有限公司保留所有权利。
Using a yeast two hybrid system and pull-down assays we demonstrate that mouse Dac (mDac) specifically binds to mouse ubiquitin-conjugating enzyme mUbc9. In contrast to a direct interaction between Drosophila dachshund (dac) and eyes absent (eya)gene products, we cannot detect by the same methods that mDac binds to mEya2, a functional mouse homologue of the Drosophila Eya. Immunostaining of various cell lines that were transfected with mDac reveals that mDac protein is found predominantly in the nucleus but translocates to the cytoplasm and condensates along the nuclear membrane in a cell-cycle dependent manner. Deletion analysis of mDac show the intracellular localization and protein stability correlates with the binding to mUbc9. The C-terminal half of mDac, which associates with mUbc9, remains cytoplasmic and is degraded in proteasome whereas the non-interacting N-terminus is exclusively nuclear and more stable than the full-length mDac or its C-terminal portion. In situ hybridization on whole-mount embryos or tissue sections detects mUbc9 transcripts in complementary and overlapping areas with mDac expression, particularly in the proliferation zone of the limb buds, the spinal cord and forebrain. Mouse embryos stained with an anti-mDac antibody document that mDac is localized both in the nucleus and the cytoplasm with a cytoplasmic predominance in migrating neural crest cells. In the proliferation zone, visible nuclear envelopes are not formed and mDac is detected throughout the cells. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.