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Evidence for a nuclear passage of nascent polypeptide-associated complex subunits in yeast

¹ Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany
² Department of Biochemistry, Humboldt University, Charité, Hessische Straße 3-4, D-10115 Berlin, Germany
³ Center for Biochemistry and Molecular Cell Chemistry, Georg-August University, Heinrich-Düker-Weg 12, D-37073 Göttingen, Germany
⁴ Franz-Volhard-Klinik, Humboldt University, Charité, Wiltbergstraße 50, D-13122 Berlin, Germany
^* Author for correspondence (e-mail: brigitte.wiedmann{at}charite.de

View larger version (47K): [in a new window]   Fig. 1. Intracellular localization of GFP fusion proteins of Egd1p, Egd2p and N11-Egd1p. (A) Expression of Egd2p-2GFP in a egd2 strain (to simulate the wild-type situation) and in the NAC-knockout strain. (B) Expression of Egd1p-2GFP in a egd1 strain (to simulate the wild-type situation) and in the NAC-knockout strain. (C) Expression of N11-Egd1p-2GFP in the NAC-knockout strain. (C) Expression of N11-Egd1p-2GFP in the NAC-knockout strain. Cells were grown to logarithmic growth phase at 30°C and stained with Hoechst 33258 to visualize nuclear DNA prior to microscopy. Positions of nuclei are indicated by arrows.

View larger version (28K): [in a new window]   Fig. 2. Subcellular localization of N-terminal truncated Egd1p proteins and co-immunoprecipitation with Egd2p. (A) Logarithmically growing wild-type cells (first two lanes), NAC-knockout cells transformed with plasmids encoding Egd2p plus Egd1p without its N-terminal 11 amino acids (N11-EGD1) or 14 amino acids (N14-EGD1), and edg1 cells (two lanes on the right) were fractionated and the cytosolic (C) and ribosomal fraction (R) were separated by SDS-PAGE. Egd1p and Egd2p were visualized by western blot analysis. The lower band in the wild-type control is most likely a degradation product. (B) Western blot analysis of immunoprecipitates from wild-type cells and the NAC-knockout strain transformed with the three indicated plasmids (label on top) with an anti-Egd1p antibody.

View larger version (28K): [in a new window]   Fig. 3. In vitro nuclear import assay of recombinant Egd1p and Egd2p. Recombinant Egd1p and Egd2p were labelled with fluorescein-5'-maleimide. Dependency of in vitro nuclear import of Egd1p on different importin proteins (left); import of Egd2p (middle); import of the standard substrate nucleoplasmin (NPL) by the different importin proteins (right). The images s, t and u represent the negative controls (import assay without importin). HeLa cells were permeabilized, the cytosol was washed out and recombinant import factors, energy mix, and labelled substrate were added. After fixation the fluorescence of the cells was observed by confocal microscopy.

View larger version (26K): [in a new window]   Fig. 4. Specificity of in vitro import of Egd2p. Various amounts of Texas Red-labelled nucleoplasmin were mixed with fluorescein-labelled Egd2p and importin 1/Rch1 and added to an in vitro import assay using permeabilized HeLa cells (a-f). Import of Egd2p is demonstrated in the presence of an equimolar amount of nucleoplasmin (a), a threefold excess (c), and a tenfold excess of nucleoplasmin (e). Images b, d and f reflect the location of nucleoplasmin in the same experiment. In g and h assays without importin and in i and j assays without energy mix are shown. The assay was performed as described above (see also Materials and Methods).

View larger version (50K): [in a new window]   Fig. 5. In vivo transport of yeast NAC proteins into nuclei is inhibited in mutants for nuclear protein import. (A) Influence of SRP1 mutations on nuclear import of N11-Egd1p-2GFP. srp1-49, srp1-31 and wild-type cells were transformed with N11-Egd1p-2GFP. Cells were grown at 25°C (right) and afterwards shifted to 37°C for 3 hours (left). Position of nuclei are indicated by arrows. (B) Influence of KAP123 and PSE1 mutations on nuclear import of N11-Egd1p-2GFP. kap123, pse1-1 and the corresponding wildtype were transformed with N11-EGD1-2GFP. Cells were grown at 25°C and stained with Hoechst 33258 prior to microscopy.

View larger version (37K): [in a new window]   Fig. 6. Identification of a domain of Egd1p responsible for nuclear import by Kap123p and Pse1p. (A) Subcellular location of the indicated N-terminal deleted Egd1 proteins in a NAC-knockout strain investigated by fluorescence microscopy. Cells were grown to logarithmic growth phase at 30°C and stained with Hoechst 33258 to visualize nuclear DNA prior to microscopy. Positions of the nuclei are indicated by arrows. (B) Influence of KAP123 and PSE1 mutations on nuclear import of N14-Egd1p-2GFP. Cells were grown at 25°C. The pse1-1 strain was shifted for 3 hours to 37°C.