The putative NLS sequence at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB.

<p>(A) The diagram shows mouse RanGAP1 wild-type (WT) (1–589) and its two C-terminal deletion mutants, CΔ23 (1–566) and CΔ49 (1–540). The mouse RanGAP1 consists of a leucine-rich repeat domain (LRR), an acidic region, and a SUMO-attachment domain (SUMO-AD) that contains a putative nuclear localization signal (NLS) (541–589) and also the lysine residue (K526) responsible for RanGAP1 SUMOylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141309#pone.0141309.ref022" target="_blank">22</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141309#pone.0141309.ref024" target="_blank">24</a>]. (B) The alignment of RanGAP homologs, including mouse RanGAP1 (P46061.2), human RanGAP1 (P46060.1), <i>Xenopus</i> RanGAP1 (O13066.1), <i>S</i>. <i>cerevisiae</i> Rna1p (P11745.2) and <i>S</i>. <i>pombe</i> Rna1p (P41391.1). (C) The NLS sequence of mouse RanGAP1 (541–589) is aligned with those of human RanGAP1 (539–587) and <i>Xenopus</i> RanGAP1 (532–580). (D-G) HeLa cells were transfected with the constructs encoding Myc-tagged RanGAP1 WT (D), CΔ23 (E), and CΔ49 (F), treated with 20 nM LMB or a control solution for 8 h, and analyzed by immunofluorescence microscopy with antibodies against RanBP2 and Myc or by immunoblotting with anti-Myc antibody (G). (H) The chart summarizes the characteristics of RanGAP1 WT, CΔ23, and CΔ49 in SUMOylation and localization in response to LMB treatment. C: Cytoplasm; N: Nucleoplasm; NPC: nuclear pore complex. Bar, 10 μm.</p

Inactivation of CRM1 by LMB causes nuclear accumulation of RanGAP1 and a loss or decrease of RanGAP1 distribution in the cytoplasm and at the NPC.

<p>(A) HeLa cells were transfected with the plasmids encoding Myc-tagged RanGAP1 for 24 h, treated with 20 nM LMB or a control solution (0.3% DMSO in PBS) for 8 h and analyzed by immunofluorescence microscopy using mouse anti-Myc mAb (9E10) and rabbit anti-RanBP2 antibodies. (B) BRL cells were treated with 20 nM LMB or a control solution for 8 h and analyzed by immunofluorescence microscopy with mouse anti-RanGAP1 mAb (19C7). (C) BRL cells were treated with either 20 nM LMB for 8 h and 16 h or a control solution for 16 h followed by immunofluorescence microscopy with antibodies specific to RanBP2 and RanGAP1. Bar, 10 μm.</p

LMB-induced nuclear accumulation of Myc-RanGAP1 is reversible after removal of LMB.

<p>(A) HeLa cells transiently expressing Myc-tagged RanGAP1 were treated with 20 nM LMB for 8 h to induce a nuclear accumulation of RanGAP1. After removal of LMB, the cells were incubated with fresh medium for the indicated times and then analyzed by immunofluorescence microscopy using anti-RanBP2 and anti-Myc antibodies. Bar, 10 μm. (B) The histogram shows the percentages of cells with the indicated N/C ratios of Myc-RanGAP1 at each time point after removal of LMB. Each bar represents the mean value ± SEM (<i>N</i> = 50, Student’s <i>t</i> test). (C) The histogram indicates the percentages of cells with (+) or without (-) NPC staining of Myc-RanGAP1 at each time point after removal of LMB (<i>N</i> = 50, Student’s <i>t</i> test). The analyses were based on three independent experiments (B and C). For each experiment, 50 cells at each time point were used for analyzing both N/C ratio and NPC staining of Myc-RanGAP1.</p

Time-course analysis of Myc-RanGAP1 redistribution during LMB treatment.

<p>(A) HeLa cells were transfected with the plasmids encoding Myc-tagged RanGAP1 for 24 h, treated with 20 nM LMB for the indicated times, and analyzed by immunofluorescence microscopy using anti-Myc (9E10) and anti-RanBP2 antibodies. Bar, 10 μm. (B) The histogram indicates the percentages of cells with the indicated N/C ratios of Myc-RanGAP1 during the time-course analysis from three independent experiments. Each bar represents the mean value ± SEM (<i>N</i> = 50, Student’s <i>t</i> test). (C) The histogram indicates the percentages of cells with (+) or without (-) NPC staining of Myc-RanGAP1 at each indicated time point (<i>N</i> = 50, Student’s <i>t</i> test). For each experiment, 50 cells at each time point were used to analyze N/C ratio and NPC staining of Myc-RanGAP1.</p

LMB treatment increases RanGAP1 SUMOylation in a RanBP2-independent manner.

<p>(A) HeLa cells were transfected with the plasmids encoding Myc-tagged RanGAP1 for 24 h, treated with 20 nM LMB or a control solution for 8 h, and analyzed by immunoblotting with mouse anti-Myc (9E10) and anti-α-tubulin mAbs. (B and C) BRL cells were treated with 20 nM LMB or a control solution for 8 h (B) and 16 h (C) followed by immunoblot analysis with mouse anti-RanGAP1 (19C7) and anti-α-tubulin mAbs. (D) HeLa cells were transfected with control or RanBP2-specific siRNA for 72 h, treated with 20 nM LMB or a control solution for 8 h, and then analyzed by immunoblotting with antibodies against RanGAP1, RanBP2 and α-tubulin.</p

RNAi-mediated depletion of CRM1 results in nuclear accumulation of RanGAP1 in mammalian cells.

<p>(A) HeLa cells were transfected with control siRNA or CRM1-specific siRNA 1 or 2 for 48 h followed by immunoblotting using antibodies specific CRM1 and α-tubulin. (B) HeLa cells were transfected with control siRNA or one of the two CRM1-specific siRNAs for 24 h and then the plasmids encoding Myc-tagged mouse RanGAP1 for 24 h followed by immunofluorescence microscopy analysis with mouse anti-Myc mAb (9E10). Bar, 10 μm. (C) The histogram shows the percentage of cells exhibiting the mean nuclear to cytoplasmic concentration ratio (N/C) of Myc-RanGAP1 in control or CRM1 RNAi cells. The immunofluorescence signal intensities of Myc-RanGAP1 in the nucleoplasm and cytoplasm were measured by ImageJ software (NIH) using cells from three independent experiments. Each bar represents the mean percentage value of cells with the indicated N/C ratio ± SEM for each treatment (<i>N</i> = 40, **<i>*P</i> < 0.001, Student’s <i>t</i> test).</p