Diversity in the Architecture of ATLs, a Family of Plant Ubiquitin-Ligases, Leads to Recognition and Targeting of Substrates in Different Cellular Environments

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20–28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress.泛素连接酶（Ubiquitin-ligases，亦称E3）是泛素-蛋白酶体系统（ubiquitin proteasome system, UPS）的组成组分，负责介导泛素向靶蛋白的转移。泛素连接酶的主要类别包含一类携带RING指结构域（RING-finger domain）的蛋白，这类蛋白在同一多肽链中包含底物识别序列，被称为单亚基RING指E3泛素连接酶。本研究聚焦于一类名为ATL的RING指E3泛素连接酶家族，该家族成员均含有跨膜结构域（transmembrane domain）与RING-H2指结构域（RING-H2 finger domain），且未携带任何其他已被报道的结构域。尽管已有研究针对拟南芥（Arabidopsis thaliana, A. thaliana）与水稻（Oryza sativa, O. sativa）中的少数ATL家族成员展开，但该家族在植物生命周期中的具体功能仍不明确。为推进该家族的功能分析研究，我们对24个植物基因组中的ATL家族成员开展了系统发育分析。结果显示，在所分析的24个植物物种中均存在ATL家族成员，其数量范围从两个基底植物物种中的20至28个，至大豆中的162个不等。对串联排列的ATL基因的分析表明，该家族基因以物种特异性的方式发生扩张。为解析ATL家族的蛋白质结构域架构，我们从1815个ATL蛋白序列中构建了75个pHMM序列标志（pHMM LOGOs），并通过酵母双杂交实验（yeast two-hybrid assays）揭示了潜在的蛋白质-蛋白质相互作用区域。研究发现，多个ATL蛋白可通过其氨基末端区域与DSK2a/泛素蛋白（DSK2a/ubiquilin）发生相互作用，提示这是一种广泛存在的相互作用模式，可能参与ATL家族的作用机制；该相互作用区域被定位至一个独特的序列标志区域。本研究不仅为ATL家族的结构域特征提供了详细信息，同时也揭示了该家族在植物中的进化与扩张模式，相关结果或可为理解植物适应环境胁迫的分子机制提供新的见解。