Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation
Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation
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DOI：
10.1046/j.1365-313x.1999.00360.x
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发表时间：
1999-01-01
期刊：
影响因子：
7.2
通讯作者：
Vierstra, RD
中科院分区：
文献类型：
--
作者：
Clough, RC;Jordan-Beebe, ET;Vierstra, RD
植物光感受器光敏色素A（phyA）从其无活性Pr形式到其生物活性Pfr形式的光转化启动其快速蛋白水解。以前的动力学和生物化学研究暗示了泛素/26 S蛋白酶体途径在这种分解中的作用，并建议涉及色蛋白内的多个结构域。为了进一步解析必需残基，我们在体外构建了一系列突变体PHY基因，并分析了转基因烟草中表达的所得光感受器的Pfr特异性降解。一个重要的位点是在多肽的C-末端一半内，因为其去除使燕麦phyA稳定为Pfr。在这一半是一组保守的赖氨酸，潜在的泛素连接所需的。这些赖氨酸的取代并不能阻止Pfr的泛素化或分解，这表明它们不是附着位点，或者在它们不存在的情况下可以使用其他赖氨酸。一个小的结构域接近的C-末端是必不可少的形式依赖的全蛋白的分解。在该结构域中仅去除6个氨基酸产生了不会迅速降解为Pfr的色蛋白。使用从马铃薯PHYA和PHYB产生的嵌合光感受器，我们发现phyA的hi末端的一半也是Pfr特异性分解所需的。只有那些含有phyA的N-末端序列的嵌合体被泛素化并迅速降解为Pfr。两者合计，我们的数据表明，而一个完整的C-末端结构域是必不可少的phyA降解，N-末端结构域是负责选择性识别和泛素化的PFR。
Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr form initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the hi-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.