Ran-dependent docking of importin-β to RanBP2/Nup358 filaments is essential for protein import and cell viability

RanBP2 captures RanGTP–importin-β complexes at cytoplasmic fibrils to ensure adequate classical NLS–mediated protein import and cell viability

Die Funktion von Nup358 im nukleocytoplasmatischen Transport

Nukleocytoplasmatischer Transport erfolgt durch in der Kernmembran verankerte Porenkomplexe. Von einigen Nukleoporinen, den Komponenten der Kernporen, wird angenommen, dass sie eine Rolle als Andockstation für Transportkomplexe auf ihrem Weg in bzw. aus dem Kern spielen können. Das Nukleoporin Nup358/RanBP2 ist der Hauptanteil der cytoplasmatischen Filamente in Vertrebratenzellen. Einige seiner Merkmale wie FG-Repeats, vier Bindestellen für die GTPase Ran und assoziiertes RanGAP1 könnten direkt mit Kerntransport in Verbindung gebracht werden. Es wurde jedoch nachgewiesen, dass der Transport von Standardkernproteinen auch ohne Nup358 erfolgen kann. Im Gegensatz zu dieser Beobachtung konnten wir zeigen, dass sich einige Kernproteine im Cytoplasma von Zellen mit reduziertem Nup358 anhäuften, darunter der putative Tumorsuppressor DBC-1 (Hutten, 2007). Des Weiteren führt die Depletion von Nup358 mittels RNA-Interferenz (RNAi) zu einer verlangsamten Transportrate von Proteinen, die entweder durch den Importin α/β-Komplex (Hutten et al., 2008) oder durch Transportin (Hutten et al., 2009) importiert werden. Somit könnte Nup358 als eine allgemeine Andockstation für spezifische Transportkomplexe fungieren. Um die Spezifität des beobachteten Effekts nach der Ausschaltung des Nukleoporins zu überprüfen, wurde eine siRNA-resistente Mutante von Nup358 in diese Zellen transfiziert, deren Menge an endogenem Nukleoporin reduziert worden war. Anwesenheit von diesem exogenen kompletten Nup358 konnte das Fehlen von endogenem Nup358 kompensieren. Wir nutzten diesen Ansatz, um spezifische Domänen von Nup358 zu eliminieren und somit Rückschlüsse auf die verantwortlichen Regionen des Kerntransports der Nup358-abhängigen Substrate ziehen zu können. So kann auf Poren-assoziiertes RanGAP1 verzichtet werden, da eine Mutante, die RanGAP1 nicht bindet, noch Import vermitteln kann. Zudem unterscheiden sich die Regionen innerhalb von Nup358, die für Transport gebraucht werden, deutlich zwischen dem Importin α/β- und dem Transportinweg. Ein N-terminales Fragment ohne jegliche Bindestelle für Ran konnte dem Kernimport von DBC-1 und eines NLS-Reporterproteins vermitteln, beide sind Importin α/β-Substrate. So konnte Import von DBC-1 durch lösliche Nup358-Fragmente inhibiert werden, die sich über die N-terminale Region der Aminosäuren 1000-1306 erstrecken. Des Weiteren konnten wir biochemisch zeigen, dass diese Interaktion unabhängig von Kerntransportrezeptoren erfolgte. Im Gegensatz hierzu benötigen Transportinkomplexe eine andere Region von Nup358 für den Import. Unsere Daten weisen darauf hin, dass das N-terminale Drittel von Nup358 als Andockstation von Transportkomplexen des Importin α/β-Weges ausreicht. Jedoch scheinen für den Transportin-abhängigen Import weitere Regionen von Nup358 gebraucht zu werden. Der Verlust von Poren-assoziiertem RanGAP1 und den vier Ranbindestellen von Nup358 können durch die entsprechenden löslichen Proteine ausgeglichen werden

Importin 7 and Nup358 Promote Nuclear Import of the Protein Component of Human Telomerase

<div><p>In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3′end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT.</p></div

RanGTP-dependent binding of importin 7 to Nup358.

<p>A, Schematic representation of GST-Nup358 fragments (compare Fig. 4A). B, Pull-down experiment. Nup358-fragments were immobilized on beads and incubated with importin 7 in the absence or presence of Ran that had been loaded with GTP. Interacting proteins were analyzed by SDS-PAGE, followed by Coomassie-staining. Fragments 806–1133 and 806–1170 were prone to degradation. The hydrophobic FG-motifs can affect the mobility of protein fragments.</p

Analysis of potential NLSs in hTERT.

<p>A, schematic representation of GFP-tagged full-length hTERT and hTERT fragments used in this study. Putative NLSs are indicated. B, hTERT-fragments accumulate in the nucleus. HeLa cells were transfected with hTERT constructs as indicated, fixed and analyzed by confocal microscopy. C, HeLa cells were transfected with constructs coding for full-length hTERT-GFP or putative NLS-mutants, as indicated and analyzed by confocal microscopy. B, C, bars, 10 µm. D, Quantification of the results in C. Error bars show the standard deviation from the mean of three independent experiments. E, Nuclear import of wild type, GFP-tagged hTERT and two NLS-mutants was analyzed by FLIP in living cells. Cells with similar expression levels were chosen for the analysis. Examples for individual cells before and after the analysis are shown at the bottom. Asterisks depict the bleached nuclear region. The graphs (top) show the mean loss in fluorescence in three independent experiments, analyzing a total of 45 cells per condition. Error bars were omitted for clarity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088887#pone.0088887.s001" target="_blank">Fig. S1</a> for an example of a FLIP-experiment with error bars).</p

The zinc finger region of Nup358 is involved in nuclear import of hTERT-GFP.

<p>A, Schematic representation of full-length Nup358 (amino acids (aa) 1–3223) and various Nup358-fragments used as rescue constructs. cc, predicted coiled-coil region, RB1-RB4, Ran-binding domains 1–4, IR, internal repeat, M, ‘middle domain’, CY, cyclophilin domain. vertical bars, FG-motifs; asterisk, region in corresponding RNA that confers resistance to siRNA. B, HeLa cells were treated with control siRNAs (not shown; compare upper left picture for localization of hTERT-GFP in cells with high levels of endogenous Nup358) or with siRNAs to deplete Nup358, and transfected with constructs coding for hTERT-GFP and HA-tagged fragments of Nup358, as indicated. Cells were fixed and analyzed by fluorescence microscopy. Antibodies against the C-terminal region of Nup358 were used to detect the endogenous protein, whereas the anti-HA antibody was used to detect Nup358-fragments. Bars, 10 or 20 µm. C, Quantification of the results shown in B. Error bars depict the standard deviation of the mean from three independent experiments.</p

siRNA-approach to identify the import receptor for hTERT.

<p>A, HeLa cells were treated with a control siRNA or with siRNAs against importin β, transportin, importin 9 or importin 7 and transfected with plasmids coding for hTERT-GFP, myc-EZI, NES-GFP₂-M9 or NES-GFP₂-NLS, as indicated. Cells were fixed and analyzed by fluorescent microscopy. EZI was detected using an antibody against the myc-tag. Bars, 10 µm. B, Quantification of the results shown in A. Error bars depict the standard deviation from the mean of at least three independent experiments. Asterisks indicate p-values <0,02 (*) or <0,002 (**), compared to the control. C, Lysates of control cells and importin β-, importin 9- or importin 7-depleted cells were analyzed by immunoblotting. Tubulin was used as a loading control.</p

Importin 7 functions as an import receptor for hTERT.

<p>A, HeLa cells expressing hTERT-GFP that had been treated with either control siRNAs or with siRNAs against importin 7 were analyzed for the dynamics of nuclear import of the reporter protein by FLIP. The graphs show the mean loss in fluorescence in three independent experiments, analyzing a total of 45 cells per condition. Error bars were omitted for clarity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088887#pone.0088887.s001" target="_blank">Fig. S1</a> for the identical experiment with error bars). B, HEK 293 cells were transfected with constructs coding for GFP or hTERT-GFP. Cell lysates were subjected to immunoprecipitation using immobilized anti-GFP-antibodies (“GFP-trap”). Reactions contained increasing amounts (0, 10 µg/ml for GFP and 0, 2.5, 5, 10 µg/ml for hTERT-GFP) of RanQ69L that had been preloaded with GTP. Precipitated proteins (GFP, hTERT-GFP and importin 7) were analyzed by immunoblotting. The weak signal for importin 7 (imp 7) in the upper panel results from the initial detection of the import receptor on the same blot (lower panel). C-F, Overexpression of exogenous importin 7 rescues nuclear import of hTERT-GFP. HeLa cells were treated with control siRNAs or with siRNAs against importin 7 and transfected with plasmids coding for either myc-EZI (C, D) or hTERT-GFP (E, F), alone or together with a plasmid coding for HA-importin 7, as indicated. Cells were fixed and analyzed by fluorescence microscopy. myc-EZI and HA-importin 7 were detected using specific antibodies against the myc- and HA-tag, respectively. Bars, 10 µm or 20 µm (control in C). D, F, Quantification of the results shown in C and E. Error bars depict the standard deviation from the mean of at least three independent experiments.</p

Model for importin 7- and Nup358-assisted import of hTERT.

<p>Cytoplasmic hTERT (TERT) interacts with Nup358, the major component of the cytoplasmic filaments of the NPC (blue rods). Upon binding of importin 7, hTERT is translocated across the NPC. In the nucleus, several mechanisms could promote the dissociation of the import complex: binding of RanGTP (R•GTP), which is generated in the vicinity of chromatin-bound RCC1, to importin 7; binding of hTERT to DNA; interaction of TERC, the RNA-component of telomerase with hTERT; accessory factors (XY) that bind to hTERT. The resulting importin 7/RanGTP complex then recycles back to the cytoplasmic side of the NPC, where it is captured by the Ran-binding sites of Nup358 (two of them are shown as red boxes). Finally, Nup358-associated RanGAP promotes GTP-hydrolysis on Ran, resulting in free RanGDP (R•GDP) and importin 7, which is kept in the vicinity of the NPC to initiate a new round of import.</p