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.