Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains

Abstract

-M.Mingot contributed equally to this work Many nuclear transport pathways are mediated by importin b-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impb and their respective basic import substrates. The Impb±Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and suf®cient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins. Keywords: chaperone/histone/importin/ribosomal protein IntroductionThe nuclear envelope (NE) divides eukaryotic cells into a nuclear and a cytoplasmic compartment, uncouples transcription from translation and thereby necessitates nucleocytoplasmic transport (reviewed in Mattaj and Englmeier, 1998;Nakielny and Dreyfuss, 1999). tRNAs, mRNAs and rRNAs, for example, need to be exported from the nucleus to the cytoplasm, where they mediate translation. Conversely, virtually all nuclear functions are dependent on proteins which are imported from the cytoplasm. The nuclear compartment already represents the ®nal destination for many import substrates, such as histones or polymerases. Numerous other proteins, however, pass through nuclei only transiently. Ribosomal proteins, for example, are ®rst imported, assemble in the nucleoli with rRNAs, and ®nally become exported as ribosomal subunits to the cytoplasm (for a review see Venema and Tollervey, 1999).Macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is normally receptor mediated. Impb-type transport receptors account for most, but not all, nuclear transport pathways (for reviews see Mattaj and Englmeier, 1998;Nakielny and Dreyfuss, 1999;Conti and Izaurralde, 2001). They constitute a diverse protein superfamily and occur in two forms, import mediators (importins) and exportins. They circulate between nucleus and cytoplasm, recognize cargo molecules and transfer them from one side of the NE to the other. Substrate loading and release is guided by a gradient of RanGTP across the NE, whereby a high nuclear RanGTP concentration favours cargo loading onto exportins and substrate displacement from importins, while cytoplasmic conditions with low RanGTP levels release substrates from exportins but allow importin±cargo complexes to fo...

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).…”

“…Gorlich and colleagues (Jakel et al, 2002) have suggested that importin 7 and importin 8, among other import receptors, may bind exposed basic regions and act as cytoplasmic chaperones to prevent aggregation; conceivably, importin 7 and importin 8 might play a role in maintaining GR solubility during nuclear transport. It will be interesting to determine if importin 7 can import other proteins that carry NLSs similar to those on NL1 and the SV40 T-antigen (Fig- ure 1A), which are considered substrates of importin ␣.…”

The Requirement of H1 Histones for a Heterodimeric Nuclear Import Receptor

“…This indicates that heterodimerization of Imp␤ with Imp7 is required for H1 import and that the simultaneous presence of both import receptors is not sufficient for import. Recently it has been shown that nuclear import of two ribosomal proteins (rpL4 and rpL6) also depends on this complex (26). However, the requirement for heterodimerization of the two import factors, as we have demonstrated here for H1 histones, has not been proven.…”

“…However, in addition to the results shown here, a growing body of evidence indicates that the Imp␤-Imp7 dimer is required as a chaperone and import receptor for nuclear proteins with extended very basic domains. A recent paper published by Jä kel et al (26) reports that import factors can prevent cytoplasmic aggregation of very basic proteins. The observation that the in vitro reconstitution of nucleosomes from isolated histones and DNA need molecular chaperones, called nucleosomal assembly proteins, and the fact that under physiological salt concentrations and without such factors, histones and DNA form aggregates and precipitate (31), also support the chaperone function of the Imp␤-Imp7 dimer.…”

“…An exception is the nuclear transport of linker histone H1, which depends on a heterodimeric receptor consisting of two members of the importin ␤-family, namely Imp␤ and Imp7 (9). This complex is also a functional import receptor of two ribosomal proteins, namely rpL4 and rpL6 (26).…”