Laboratorio de Biotecnología Molecular de Plantas, División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C, San Luis Potosí, SLP, Mexico.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Cell Stress Chaperones. 2021 Mar;27(2):165-176. doi: 10.1007/s12192-022-01261-5. Epub 2022 Feb 16.
The Arabidopsis thaliana glycine-rich domain protein 2 (AtGRDP2) gene encodes a protein of unknown function that is involved in plant growth and salt stress tolerance. The AtGRDP2 protein (787 aa, At4g37900) is constituted by three domains: a DUF1399 located at the N-terminus, a potential RNA Recognition Motif (RRM) in the central region, and a short glycine-rich domain at the C-terminus. Herein, we analyzed the subcellular localization of AtGRDP2 protein as a GFP translational fusion and found it was localized in the cytosol and the nucleus of tobacco leaf cells. Truncated versions of AtGRDP2 showed that the DUF1399 or the RRM domains were sufficient for nuclear localization. In addition, we performed a yeast two-hybrid split-ubiquitin assay (Y2H) to identify potential interactors for AtGRDP2 protein. The Y2H assay identified proteins associated with RNA binding functions such as PABN3 (At5g65260), EF-1α (At1g07920), and CL15 (At3g25920). Heterodimeric associations in planta between AtGRDP2 and its interactors were carried out by Bimolecular Fluorescence Complementation (BiFC) assays. The data revealed heterodimeric interactions between AtGRDP2 and PABN3 in the nucleus and AtGRDP2 with EF-1α in the cytosol, while AtGRDP2-CL15 associations occurred only in the chloroplasts. Finally, functional characterization of the protein-protein interaction regions revealed that both DUF1399 and RRM domains were key for heterodimerization with its interactors. The AtGRDP2 interaction with these proteins in different compartments suggests that this glycine-rich domain protein is involved in post-transcriptional processes.
拟南芥甘氨酸丰富域蛋白 2（AtGRDP2）基因编码一种未知功能的蛋白质，参与植物生长和耐盐胁迫。AtGRDP2 蛋白（787 个氨基酸，At4g37900）由三个结构域组成：N 端的 DUF1399 结构域、中心区域的潜在 RNA 识别基序（RRM）和 C 端的短甘氨酸丰富结构域。本文分析了 AtGRDP2 蛋白的亚细胞定位作为 GFP 翻译融合蛋白，并发现其定位于烟草叶片细胞的细胞质和细胞核中。AtGRDP2 的截短版本表明，DUF1399 或 RRM 结构域足以进行核定位。此外，我们进行了酵母双杂交分裂泛素测定（Y2H）以鉴定 AtGRDP2 蛋白的潜在相互作用蛋白。Y2H 测定鉴定了与 RNA 结合功能相关的蛋白，如 PABN3（At5g65260）、EF-1α（At1g07920）和 CL15（At3g25920）。通过双分子荧光互补（BiFC）测定在体内进行了 AtGRDP2 与其相互作用蛋白之间的异二聚体关联。数据显示，AtGRDP2 与 PABN3 在核内以及 AtGRDP2 与 EF-1α 在细胞质中形成异二聚体相互作用，而 AtGRDP2-CL15 相互作用仅发生在叶绿体中。最后，对蛋白-蛋白相互作用区域的功能表征表明，DUF1399 和 RRM 结构域对于与相互作用蛋白的异二聚化都是关键的。AtGRDP2 与这些蛋白在不同隔室中的相互作用表明，这种富含甘氨酸的结构域蛋白参与了转录后过程。
