Stefan Jäkel

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

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.

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin β 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 β (Impβ) 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 Impβ/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impβ together with Imp7. Import by the Imp7/Impβ 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 Impβ and Imp7, whereby the two Ran‐binding sites act synergistically. However, whereas an Impβ/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 Impβ. Within the Impβ/Imp7 heterodimer, however, Imp7 plays a more passive role than Impβ and resembles an import adapter.

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

Many nuclear transport pathways are mediated by importin β‐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 Impβ and their respective basic import substrates. The Impβ–Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient 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.

Mitogen‐activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment

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 Mnk2D228G–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.