Importin 13: a novel mediator of nuclear import and export

Abstract

Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO-1/sentrin-conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin beta-like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Fig. 1. Identification of potential Imp13-specific transport substrates. Immobilized Imp13 was used to fish interacting proteins from a cytosolic HeLa extract. Binding was performed in the absence or presence of RanQ69L GTP (5 µM) to identify potential import or export substrates, respectively. Bound proteins were separated by SDS–PAGE, visualized by Coomassie staining, digested with trypsin and finally identified by peptide sequencing and mass spectrometry. The following proteins were identified: RL5, ribosomal protein L5 (accession No. P46777); RBM8 (Y14), RNA-binding motif protein 8 (AAF37551); NF-YB-like protein (AAF67146); hUBC9 (AAH00427); MGN, Mago Nashi protein homologue (P50606); NPAP, nuclear pore-associated protein (AAD53401); eIF1A, eukaryotic translation initiation factor 1A (P47813).

Fig. 2. hUBC9 behaves like an Imp13-specific import substrate. (A) Immobilized hUBC9 was used to bind nuclear transport receptors from a cytoplasmic HeLa cell extract. Starting material and bound fractions were analysed by SDS–PAGE followed by Coomassie staining. Note, Imp13 was bound specifically to the matrix and this binding was abolished by the GTP-bound form of RanQ69L (5 µM) that was used to mimic a nuclear environment. (B) The samples from (A) were analysed by immunoblotting with various antibodies. Imp13 was highly enriched in the hUBC9-bound fraction, while binding of other transport receptors was insignificant. (C) Binding to hUBC9 was performed as in (A), except that an E.coli lysate with recombinant Imp13 was used as a source of nuclear transport receptors. The recombinant receptor behaved like the native protein from the HeLa extract (see A).

Fig. 3. Imp13 mediates import of hUBC9. Import of fluorescently labelled GST–hUBC9 (1 µM) into nuclei of permeabilized cells was performed for 5 min with the indicated combinations of nuclear transport receptors (1 µM each). Panels show confocal sections of the hUBC9 distribution after import and fixation. Nuclear hUBC9 accumulation was dependent on the presence of Imp13, Ran mix and an energy-regenerating system (see Materials and methods).

Fig. 4. Imp13 mediates import of the RBM8–MGN complex. (A) An E.coli lysate with recombinant Imp13 was used as the starting material for the binding experiment. Imp13 bound very efficiently and in a RanGTP-sensitive manner to immobilized RBM8 and the RBM8–MGN complex. (B) Binding was performed as in (A), except that MGN was immobilized instead of RBM8. Imp13 bound only weakly to MGN alone, but efficiently to the MGN–RBM8 complex. (C) A pre-formed RBM8–MGN complex was fluorescently labelled and used at 1 µM as an import substrate as described in Figure 3. Import was efficient with Imp13 and hardly detectable with any other nuclear transport receptor (each used at 2 µM).

Fig. 5. Imp13 mediates nuclear export of eIF1A. (A) Fluorescent GST–eIF1A (1 µM) was allowed to diffuse for 6 min into nuclei. One sample was fixed immediately. The others were incubated with the indicated additions for a further 5 min before they were also fixed. The presence of Imp13 (2 µM), Ran and energy resulted in efficient eIF1A export and complete loss of the nucleolar signal. (B) A comparison of the indicated combinations of nuclear transport receptors (2 µM) in their export activity towards eIF1A. Export was performed as in (A).

Fig. 6. Characterization of the eIF1A–Imp13 interaction. (A) Binding of nuclear transport receptors from a HeLa cell extract to immobilized eIF1A was analysed by immunoblotting with the indicated antibodies. Imp13 was highly enriched in the bound fraction, provided RanQ69L (5 µM) had been added to the incubation. (B) Imp13 was bound from an E.coli lysate to immobilized RanQ69L (GTP). This binding was antagonized efficiently by the import substrate GST–hUBC9. In contrast, GST–eIF1A did not compete the Imp13–Ran interaction and instead engaged in a trimeric eIF1A–Imp13–RanGTP complex. GST–hUBC9 and GST–eIF1A were each used at 10 µM (monomers) which compares with ∼1 µM Imp13 present in the extract. (C) Recombinant Imp13 was bound to immobilized eIF1A in the presence of the indicated combinations of RanQ69LGTP and GST–hUBC9. RanGTP engaged in trimeric eIF1A–Imp13–Ran complexes, but otherwiese had no effect on the Imp13–eIF1A interaction. hUBC9 efficiently competed Imp13 binding to eIF1A, but the presence of RanGTP fully abrogated this effect.

Fig. 7. Scheme for the interactions of Imp13 with RanGTP, its export substrate eIF1A and an import cargo. Under cytoplasmic conditions, RanBP1/RanBP2 and RanGAP remove RanGTP from Imp13. This is insufficient to release eIF1A from Imp13, but allows a displacement of eIF1A by import substrates. The Imp13–import substrate complex can then enter nuclei. In a nuclear environment, the import substrate is displaced by RanGTP, which in turn favours the eIF1A–Imp13 interaction and thereby allows eIF1A export.