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

He S, Huang K, Zhang X, Yu X, Huang P, An C - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.

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.

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The NLS of PsLSD1 is involved in interacting with importin α.(A) PsLSD1 interacts with AtIMPα1, AtIMPα2, AtIMPα3, and AtIMPα1 in yeast. pGBK-AtIMPα1, pGBK-AtIMPα2, pGBK-AtIMPα3, pGBK-AtIMPα4, and pGBKT7 were co-transformed with pGAD-PsLSD1 into yeast AH109 respectively, and β-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 AtIMPα1 in vitro. Purified MBP-PsLSD1 was incubated with GST or GST-AtIMPα1 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 AtIMPα1 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 µm.
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pone-0022131-g003: The NLS of PsLSD1 is involved in interacting with importin α.(A) PsLSD1 interacts with AtIMPα1, AtIMPα2, AtIMPα3, and AtIMPα1 in yeast. pGBK-AtIMPα1, pGBK-AtIMPα2, pGBK-AtIMPα3, pGBK-AtIMPα4, and pGBKT7 were co-transformed with pGAD-PsLSD1 into yeast AH109 respectively, and β-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 AtIMPα1 in vitro. Purified MBP-PsLSD1 was incubated with GST or GST-AtIMPα1 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 AtIMPα1 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 µm.

Mentions: To elucidate how the NLS of PsLSD1 is involved in the nuclear import of PsLSD1, we analyzed the possible interaction between the NLS of PsLSD1 and nuclear import protein importin α, which recognizes the classical NLSs. The Arabidopsis genome contains nine genes encoding importin α isoforms [21]. We initially analyzed the interactions between four of these importin α isoforms (AtIMPα1, AtIMPα2, AtIMPα3, and AtIMPα4) and PsLSD1. Yeast two-hybrid assay showed that all these four importin α proteins associated with PsLSD1 (Figure 3A). To confirm the interaction between PsLSD1 and importin α, we performed in vitro GST pull-down assay. Very little MBP-PsLSD1 protein was pulled-down by GST-bound beads, whereas significantly more MBP-PsLSD1 protein was pulled-down by GST-AtIMPα1-bound beads (Figure 3B), indicating that PsLSD1 binds to AtIMPα1 in vitro.


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

He S, Huang K, Zhang X, Yu X, Huang P, An C - PLoS ONE (2011)

The NLS of PsLSD1 is involved in interacting with importin α.(A) PsLSD1 interacts with AtIMPα1, AtIMPα2, AtIMPα3, and AtIMPα1 in yeast. pGBK-AtIMPα1, pGBK-AtIMPα2, pGBK-AtIMPα3, pGBK-AtIMPα4, and pGBKT7 were co-transformed with pGAD-PsLSD1 into yeast AH109 respectively, and β-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 AtIMPα1 in vitro. Purified MBP-PsLSD1 was incubated with GST or GST-AtIMPα1 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 AtIMPα1 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 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139611&req=5

pone-0022131-g003: The NLS of PsLSD1 is involved in interacting with importin α.(A) PsLSD1 interacts with AtIMPα1, AtIMPα2, AtIMPα3, and AtIMPα1 in yeast. pGBK-AtIMPα1, pGBK-AtIMPα2, pGBK-AtIMPα3, pGBK-AtIMPα4, and pGBKT7 were co-transformed with pGAD-PsLSD1 into yeast AH109 respectively, and β-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 AtIMPα1 in vitro. Purified MBP-PsLSD1 was incubated with GST or GST-AtIMPα1 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 AtIMPα1 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 µm.
Mentions: To elucidate how the NLS of PsLSD1 is involved in the nuclear import of PsLSD1, we analyzed the possible interaction between the NLS of PsLSD1 and nuclear import protein importin α, which recognizes the classical NLSs. The Arabidopsis genome contains nine genes encoding importin α isoforms [21]. We initially analyzed the interactions between four of these importin α isoforms (AtIMPα1, AtIMPα2, AtIMPα3, and AtIMPα4) and PsLSD1. Yeast two-hybrid assay showed that all these four importin α proteins associated with PsLSD1 (Figure 3A). To confirm the interaction between PsLSD1 and importin α, we performed in vitro GST pull-down assay. Very little MBP-PsLSD1 protein was pulled-down by GST-bound beads, whereas significantly more MBP-PsLSD1 protein was pulled-down by GST-AtIMPα1-bound beads (Figure 3B), indicating that PsLSD1 binds to AtIMPα1 in vitro.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.

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.

Show MeSH