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.

-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...

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.

No abstract

No abstract

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 A crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

No abstract