An Interactome Analysis Reveals That Arabidopsis Thaliana GRDP2 Interacts with Proteins Involved in Post-transcriptional Processes

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.