Hedhili Sabah, De Mattei Marie-Véronique, Coudert Yoan, Bourrié Isabelle, Bigot Yves, Gantet Pascal
Université François Rabelais, UFR des Sciences et Techniques, Unité sous Contrat reconnue par l'Institut National de la Recherche Agronomique, Facteurs de Transcription et Ingénierie Métabolique Végétale, Biomolécules et Biotechnologies Végétales, EA 2106, Parc de Grandmont, 37200 Tours, France.
BMC Res Notes. 2010 Nov 12;3:301. doi: 10.1186/1756-0500-3-301.
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.
CrMYC2是一种早期茉莉酸响应型bHLH转录因子，参与调控长春花中萜类吲哚生物碱生物合成途径相关基因的表达。在本文中，我们鉴定了CrMYC2核定位所必需的氨基酸结构域。
我们通过洋葱表皮细胞中的瞬时表达试验，检测了与绿色荧光蛋白（GFP）融合的完整CrMYC2及其各种缺失突变体的细胞内定位。对该蛋白的序列分析揭示了四个假定的碱性核定位信号（NLS）。试验表明，预测的NLS单独均无活性。对CrMYC2的进一步功能剖析表明，该转录因子的核定位涉及位于蛋白C端区域的三个结构域的协同作用。前两个结构域位于氨基酸残基454 - 510和510 - 562处，包含典型的碱性单部分NLS；这些区域分别称为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，在其他系统发育相关的bHLH转录因子中高度保守。我们的数据表明，这三个结构域单独时无活性，但协同作用将CrMYC2靶向细胞核。
本研究鉴定了三个协同作用将CrMYC2转录因子靶向细胞核的氨基酸结构域。对这些氨基酸结构域进行进一步的精细结构/功能分析，将有助于鉴定新的NLS结构域，并有助于研究CrMYC2 bHLH转录因子核定位所涉及的相关分子机制。
