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

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Abstract

Background: 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. Methodology/Principal Findings: 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. Conclusions/Significance: 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. © 2011 He et al.

Figures

  • Figure 1. PsLSD1 is involved in negatively regulating PCD. (A) RT-PCR analysis of the expression of AtLSD1 in the lsd1-2 mutant. AtUBQ10 was used as an internal control. (B) Western blot analysis of transgenic plant lsd1-2/HA-PsLSD1 lines using an anti-HA monoclonal antibody. Wild-type plant was used as negative control. Various transgenic lines are numbered; NC and LC represent negative control and loading control, respectively. (C) Lesion phenotypes of various plant lines after SA treatment. Leaves were photographed at 5 days post-SA spray. Each leaf shown is a representative of about 30 leaves in three independent experiments. (D) Ion leakage profiles of various plant lines after SA treatment. At 3 days postSA spray, conductivity of leaf discs from various plant lines was measured at the indicated time. SD indicates four independent data points. The experiment was performed three times with similar results. doi:10.1371/journal.pone.0022131.g001
  • Figure 2. The NLS of PsLSD1 is located in the three LSD1-type zinc finger motifs. (A) Schematic diagram of GFP-tagged PsLSD1 and its deletion mutants. zf1, zf2, and zf3 indicate the first, second, and third zinc finger motif, respectively. NTD and CTD represent the N- and C-terminal domain, respectively. (B) PsLSD1 is localized in the nucleus. (C) The N-terminal domain is sufficient and necessary for PsLSD1 nuclear localization. (D) All the three zinc finger motifs are necessary for PsLSD1 nuclear localization. (E) Tertiary structure of each zinc finger motif is essential for PsLSD1 nuclear localization. ZF1CA, ZF2CA, and ZF3CA indicate the mutation of the second cysteine to alanine within the first, second, and third zinc finger motif, respectively. (F) Basic residues of each zinc finger motif are essential for PsLSD1 nuclear localization. ZF1RA, ZF2RA, and ZF3RA indicate the mutation of the first arginine to alanine within the first, second, and third zinc finger motif, respectively. Arabidopsis mesophyll protoplasts were transfected with the indicated constructs, and samples were stained with DAPI to indicate positions of nuclei. Fluorescent images were taken at 12– 16 h after transfection. Each protoplast shown is a representative of at least twenty protoplasts in three independent experiments. Epifluorescence (left), DAPI (middle), and merged (right) images are shown. Scale bar is 20 mm. doi:10.1371/journal.pone.0022131.g002
  • Figure 3. The NLS of PsLSD1 is involved in interacting with importin a. (A) PsLSD1 interacts with AtIMPa1, AtIMPa2, AtIMPa3, and AtIMPa1 in yeast. pGBK-AtIMPa1, pGBK-AtIMPa2, pGBK-AtIMPa3, pGBK-AtIMPa4, and pGBKT7 were co-transformed with pGAD-PsLSD1 into yeast AH109 respectively, and b-galactosidase activity of the resulting clones was measured. ‘‘T+p53’’ and ‘‘T+Lam’’ are positive and negative controls for the yeast two-hybrid assay, respectively. (B) PsLSD1 directly binds to AtIMPa1 in vitro. Purified MBP-PsLSD1 was incubated with GST or GST-AtIMPa1 bound to glutathione particles. Pulled-down proteins and ‘‘Input’’ sample (purified MBP-PsLSD1) were detected by Western blot using an anti-MBP polyclonal antibody. (C) PsLSD1 interacts with AtIMPa1 in vivo. YN-AtIMPa1 was co-transfected with YC and YC-PsLSD1 into Arabidopsis mesophyll protoplasts, respectively, and samples were stained with DAPI to indicate positions of nuclei. Fluorescent images were taken at 12–16 h after transfection. Each protoplast shown is a representative of at least twenty protoplasts in two independent experiments. BF indicates Bright Field, and scale bar is 20 mm. doi:10.1371/journal.pone.0022131.g003
  • Figure 4. The NLS of PsLSD1 associates with AtIMPa1 in vitro. Purified MBP-PsLSD1, MBP, MBP-PsLSD1NTD, MBP-PsLSD1CTD, MBPPsLSD1DZF1, MBP-PsLSD1DZF2, and MBP-PsLSD1DZF3 were incubated with GST-AtIMPa1 or GST (control) bound to MagneGSTTM glutathione particles, respectively. Pulled-down proteins and ‘‘Input’’ sample (purified MBP fusion proteins) were detected by Western blot using an anti-MBP polyclonal antibody. The experiments were performed two times with similar results. doi:10.1371/journal.pone.0022131.g004
  • Figure 5. The three LSD1-type zinc finger motifs of LSD1s are highly conserved. The LSD1s are shown in the following order: Pisum sativa (Ps) LSD1 (GenBank accession number: AAY40471.1), Glycine max (Gm) LSD1 (UniProtKB accession number: C6TGF7), Vitis vinifera (Vv) LSD1 (UniProtKB accession number: A7QC17), Brassica oleracea (Bo) LSD1 (UniProtKB accession number: Q8W195), and Arabidopsis thaliana (At) LSD1 (UniProtKB accession number: P94077). Amino acid sequences of LSD1s were analyzed by the ClustalW2 program (http://www.ebi.ac.uk/Tools/ clustalw2/index.html). The three conserved zinc finger motifs are underlined. The zinc chelating cysteine residues and conserved basic residues within the zinc finger motifs are shown in the upper region of the sequences. doi:10.1371/journal.pone.0022131.g005

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He, S., Huang, K., Zhang, X., Yu, X., Huang, P., & An, C. (2011). The LSD1-type zinc finger motifs of pisum sativa LSD1 are a novel nuclear localization signal and interact with importin Alpha. PLoS ONE, 6(7). https://doi.org/10.1371/journal.pone.0022131

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