Importin beta , transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells

Abstract

The assembly of eukaryotic ribosomal subunits takes place in the nucleolus and requires nuclear import of ribosomal proteins. We have studied this import in a mammalian system and found that the classical nuclear import pathway using the importin α/β heterodimer apparently plays only a minor role. Instead, at least four importin β-like transport receptors, namely importin β itself, transportin, RanBP5 and RanBP7, directly bind and import ribosomal proteins. We found that the ribosomal proteins L23a, S7 and L5 can each be imported alternatively by any of the four receptors. We have studied rpL23a in detail and identified a very basic region to which each of the four import receptors bind avidly. This domain might be considered as an archetypal import signal that evolved before import receptors diverged in evolution. The presence of distinct binding sites for rpL23a and the M9 import signal in transportin, and for rpL23a and importin α in importin β might explain how a single receptor can recognize very different import signals.

Importin β3 mediates the nuclear import of human ribosomal protein L7 through its interaction with the multifaceted basic clusters of L7

“…3A). This weak signal is probably due to the digitonin treatment as reported previously [13]. Nevertheless, when the import experiment was carried out with a cell lysate prepared from HeLa cells, the same heavy nuclear accumulation of fluorescent L7 signal was clearly observed (Fig.…”

“…To clarify the role of importin b3 as a carrier protein for nuclear import of L7, we carried out an ex vivo nuclear uptake assay using digitonin-treated HeLa cells [13] and fluorescently labelled L7. The fluorescence signal accumulated in the nucleoli (Fig.…”

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Similarly, the BIB domain and the GR 407-525 fragment of GR bound to importin 7 in a HeLa cell extract ( Figure 3D, lanes 4 and 5), and mutation of GR 407-525 K513-515A again abrogated this interaction ( Figure 3D, lane 6). We conclude that GR can bind importin 7 and importin 8.Finally, consistent with other importin 7 and importin 8 substrates (Jakel and Gorlich, 1998;Dean et al, 2001;Jakel et al, 2002), the presence of a GTPase-deficient Ran mutant, RanQ69L, reduced the affinity of these import receptors for in vitro-transcribed and translated full-length GR ( Figure 3E, lanes 5 and 7), suggesting that these importins may import GR. In addition, RanQ69L reduced the binding observed between GST-importin ␣ and in vitro-transcribed and translated full-length GR ( Figure 3E, lane 3).…”

“…Plasmid pGST-GFP-407-525 K513-515A was made from pGST-GFP-407-525 by Quick Change Mutagenesis. pGST-GFP-BIB was constructed by amplifying the DNA encoding the BIB domain from plasmid 4zL123a-cys (Jakel and Gorlich, 1998) using primers containing EcoRI and XhoI sites. Plasmid pGEX-2t-GFP-NLS was generously provided by Mary S. Moore (Baylor College of Medicine, Houston, TX).…”

“…Importin 7 was purified as in Jakel and Gorlich (1998), and importin 8 was purified as in Dean et al (2001) with variations. Proteins were expressed in XL1-Blue cells (Stratagene), induced with 4 mM IPTG for 8 h at 16°C at an OD595 of 0.8.…”