Interaction Between Methyl CpG-Binding Protein and Ran GTPase during Cell Division in Tobacco Cultured Cells

Abstract

Methyl CpG-binding proteins are considered to play critical roles in epigenetic control of gene expression by recognizing and interacting with 5-methylcytosine (m(5)C) in eukaryotes. However, among 13 corresponding genes in Arabidopsis thaliana, designated as featuring a methyl-binding domain (MBD), only four have so far been shown actually to bind to m(5)C. One example, AtMBD5, was selected here to screen for interacting proteins. Yeast two-hybrid assays were used for screening, and physical interaction was confirmed by pull-down and bimolecular fluorescence complementation (BiFC) assays. Cellular localization was analysed by fluorescence-tagged fusion proteins using tobacco (Nicotiana tabacum) cultured bright yellow 2 cells. A gene finally identified was found to encode AtRAN3, a protein that belongs to the Ran GTPase family, which plays a critical role in nucleocytoplasmic transport and spindle bipolarization during cell division. AtMBD5 and AtRAN3 were clearly shown to interact in the nucleus by BiFC. On co-expression of AtMBD5-cyan fluorescence protein and yellow fluorescence protein-AtRAN3 in tobacco cells, both localized to the nucleus in the resting stage, migrating to the cytoplasm, primarily around chromatin, during mitosis, particularly at metaphase. These results suggest that AtMBD5 becomes localized to the vicinity of chromosomes with the aid of AtRAN3 during cell division, and may play an important role not only in maintenance of chromatin structures by binding to m(5)C, but also in progress through mitosis by detaching from m(5)C. The present findings also shed light on the physiological function of Ran GTPases, direct target proteins of which have not thus far been well defined, suggesting their key role in chromatin movements in plant cells.