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.
Highlights
The nuclear envelope (NE) is a structure that lies between the nucleoplasm and cytoplasm
Fluorescence of GST-enhanced GFP (EGFP) was intense around the nucleus, perhaps due to stacked endoplasmic reticulum (ER) membranes and a dense cytoplasm
NLSb1 was found in the rod domain, while other nuclear localization signal (NLS) resided in the tail domain
Summary
The nuclear envelope (NE) is a structure that lies between the nucleoplasm and cytoplasm. The nuclear lamina is a proteinaceous meshwork composed mainly of the type V intermediate filament proteins, the nuclear lamins, which line the inner surface of the nuclear membranes. In addition to their role in diffusing local mechanical stress on the NE, lamins are involved in many aspects of nuclear function (Gruenbaum et al, 2003, 2005; Dahl et al, 2004; Starr and Fischer, 2005; Dechat et al, 2010). A mutation in NLSm4 was introduced using the primers NLS4mt-F and NLS4mt-R, and in YNL using YNLmt-F and YNLmt-R
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