Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure

• Published in: Nucleus (Austin, Tex.) Vol. 8; no. 3; pp. 312 - 322
• Main Authors: Mochizuki, Ryota, Tsugama, Daisuke, Yamazaki, Michihiro, Fujino, Kaien, Masuda, Kiyoshi
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 04.05.2017
• Subjects: actin; chromatin; Daucus carota - cytology; Daucus carota - metabolism; lamina-like structure; Nuclear Lamina - metabolism; nuclear membrane; Original Research; Plant Proteins - chemistry; Plant Proteins - metabolism; plants; Protein Binding; Protein Domains; Protein Transport; protein-protein interaction; transcription factor; lamina-like structure; transcription factor; actin; plants; protein-protein interaction; chromatin; nuclear membrane
• ISSN: 1949-1034; 1949-1042
• DOI: 10.1080/19491034.2017.1280210

NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.