Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor.
Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor.
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DOI：
10.1186/1756-0500-3-301
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发表时间：
2010-11-12
影响因子：
1.8
通讯作者：
Gantet P
中科院分区：
文献类型：
--
作者：
Hedhili S;De Mattei MV;Coudert Y;Bourrié I;Bigot Y;Gantet P
CrMYC2是茉莉酸反应的早期转录因子，参与调控长春花萜烯类吲哚生物碱生物合成途径基因的表达。在本文中，我们确定了CrMYC2核靶向所需的氨基酸结构域。我们用洋葱表皮细胞的瞬时表达实验检测了完整的CrMYC2和各种缺失突变体的细胞内定位，这些突变体都与GFP融合。对该蛋白的序列分析表明，该蛋白存在四个假定的基本核定位信号(NLS)。分析表明，没有一个预测的NLS是单独活跃的。对CrMYC2的进一步功能分析表明，该转录因子的核靶向涉及位于蛋白质C-末端的三个结构域的协同作用。前两个结构域分别位于氨基酸残基454-510和510-562，含有基本的经典单组分NLSS，分别称为NLS3(KRPRKR)和NLS4(EAERQRREK)。第三个结构域位于残基617和652之间，富含碱性氨基酸，这些氨基酸在其他系统发育相关的bHLH转录因子中非常保守。我们的数据表明，这三个结构域在分离时是不活跃的，但协同作用将CrMYC2靶向细胞核。本研究确定了三个氨基酸结构域，它们协同作用将CrMYC2转录因子靶向细胞核。对这些氨基酸结构域的进一步精细结构/功能分析将有助于鉴定新的NLS结构域，并将有助于研究与CrMYC2 bHLH转录因子核靶向相关的分子机制。
CrMYC2 is an early jasmonate-responsive bHLH transcription factor involved in the regulation of the expression of the genes of the terpenic indole alkaloid biosynthesis pathway in Catharanthus roseus. In this paper, we identified the amino acid domains necessary for the nuclear targeting of CrMYC2. We examined the intracellular localization of whole CrMYC2 and of various deletion mutants, all fused with GFP, using a transient expression assay in onion epidermal cells. Sequence analysis of this protein revealed the presence of four putative basic nuclear localization signals (NLS). Assays showed that none of the predicted NLS is active alone. Further functional dissection of CrMYC2 showed that the nuclear targeting of this transcription factor involves the cooperation of three domains located in the C-terminal region of the protein. The first two domains are located at amino acid residues 454-510 and 510-562 and contain basic classical monopartite NLSs; these regions are referred to as NLS3 (KRPRKR) and NLS4 (EAERQRREK), respectively. The third domain, between residues 617 and 652, is rich in basic amino acids that are well conserved in other phylogenetically related bHLH transcription factors. Our data revealed that these three domains are inactive when isolated but act cooperatively to target CrMYC2 to the nucleus. This study identified three amino acid domains that act in cooperation to target the CrMYC2 transcription factor to the nucleus. Further fine structure/function analysis of these amino acid domains will allow the identification of new NLS domains and will allow the investigation of the related molecular mechanisms involved in the nuclear targeting of the CrMYC2 bHLH transcription factor.