title: Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain.
abstract: Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.
authors.affiliation: Laboratory of Plant Functional Biology, Chair of Botany and Agronomy, Graduate School of Agriculture, Hokkaido University Hokkaido, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Laboratory of Crop Physiology, Chair of Botany and Agronomy, Graduate School of Agriculture, Hokkaido University Hokkaido, Japan.
authors.forename: Kaien
authors.initials: K
authors.lastname: Fujino
authors.affiliation: Laboratory of Plant Functional Biology, Chair of Botany and Agronomy, Graduate School of Agriculture, Hokkaido University Hokkaido, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Ogawa
authors.affiliation: Laboratory of Plant Functional Biology, Chair of Botany and Agronomy, Graduate School of Agriculture, Hokkaido University Hokkaido, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Masuda
keywords: Daucus carota; GFP-fusion protein; NMCP1; lamin; nuclear envelope; nuclear localization signal (NLS); site-directed mutation