The LSD1-type zinc finger motifs of Pisum sativa LSD1 are a novel nuclear localization signal and interact with importin alpha

• Published in: PloS one 2011-07, Vol.6 (7), p.e22131
• Main Authors: He, Shanping, Huang, Kuowei, Zhang, Xu, Yu, Xiangchun, Huang, Ping, An, Chengcai
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Amino Acids, Basic - metabolism; Analysis; Apoptosis; Arabidopsis; Arabidopsis - metabolism; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biology; Cell death; Cell Nucleus - metabolism; Clonal deletion; Cloning; Conserved Sequence - genetics; Deletion mutant; Deoxyribonucleic acid; DNA; DNA binding proteins; Fluorescence; Gene expression; Green fluorescent protein; Homology; Karyopherins - metabolism; Laboratories; Life sciences; Localization; Molecular Sequence Data; Mutagenesis; Mutants; Nuclear Localization Signals - chemistry; Nuclear Localization Signals - metabolism; Nuclear transport; Nuclei; Pea; Pisum sativum - cytology; Pisum sativum - metabolism; Plant Proteins - chemistry; Plant Proteins - metabolism; Protein Binding; Protein structure; Protein Structure, Tertiary; Protein Transport; Protein-protein interactions; Proteins; Site-directed mutagenesis; Structure-Activity Relationship; Studies; Tertiary structure; Transcription; Transcription factors; Transgenic plants; Yeast; Zinc; Zinc finger proteins; Zinc Fingers
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0022131

Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD). Arabidopsis thaliana LSD1 (AtLSD1) contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1), which is a homolog of AtLSD1. Subcellular localization analysis of green fluorescent protein (GFP)-tagged PsLSD1 indicates that PsLSD1 is localized in the nucleus. Using a series of GFP-tagged PsLSD1 deletion mutants, we found that the three LSD1-type zinc finger motifs of PsLSD1 alone can target GFP to the nucleus, whereas deletion of the three zinc finger motifs or any individual zinc finger motif causes PsLSD1 to lose its nuclear localization, indicating that the three zinc finger motifs are necessary and sufficient for its nuclear localization. Moreover, site-directed mutagenesis analysis of GFP-tagged PsLSD1 indicates that tertiary structure and basic amino acids of each zinc finger motif are necessary for PsLSD1 nuclear localization. In addition, yeast two-hybrid, pull-down, and BiFC assays demonstrate that the three zinc finger motifs of PsLSD1 directly bind to importin α in vitro and in vivo. Our data demonstrate that the LSD1-type zinc finger motifs of PsLSD1 are a novel nuclear localization signal and directly bind to importin α, and suggest that the nuclear import of LSD1 may rely on the interaction between its zinc finger motifs and importin α. Moreover, the nuclear localization of PsLSD1 suggests that LSD1 may function as a transcription regulator involved in negatively regulating PCD.