Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm

Abstract

Importin beta-type transport receptors mediate the vast majority of transport pathways between cell nucleus and cytoplasm. We identify here the translation elongation factor 1A (eEF1A) as the predominant nuclear export substrate of RanBP21/exportin 5 (Exp5). This cargo-exportin interaction is rather un usual in that eEF1A binds the exportin not directly, but instead via aminoacylated tRNAs. Exp5 thus represents the second directly RNA-binding exportin and mediates tRNA export in parallel with exportin-t. It was suggested recently that 10-15% of the cellular translation would occur in the nucleus. Our data rule out such a scenario and instead suggest that nuclear translation is actively suppressed by the nuclear export machinery. We found that the vast majority of translation initiation factors (eIF2, eIF2B, eIF3, eIF4A1, eIF5 and eIF5B), all three elongation factors (eEF1A, eEF1B and eEF2) and the termination factor eRF1 are strictly excluded from nuclei. Besides Exp5 and importin 13, CRM1 and as yet unidentified exportins also contribute to the depletion of translation factors from nuclei.

Fig. 1. (A) Identification of the eEF1A–tRNA complex as a putative export substrate for Exp5. A cytosolic extract from HeLa cells was depleted of endogenous nuclear transport receptors and 500 µl aliquots were then each supplemented with a single nuclear transport receptor (1 µM) as indicated. Export complexes were formed with 2 µM zz-tagged RanGTP (GTPase-deficient RanQ69L mutant) and purified with IgG–Sepharose. Analysis was by SDS–PAGE followed by Coomassie staining. The load of the bound fractions corresponds to 40 times that of the starting material. With Exp5, a stoichiometric 50 kDa band was recovered and identified by peptide fingerprinting as eEF1A. (B) Export complexes were formed with the indicated transport receptors as in (A), but analysis was by immunoblotting with antibodies against the indicated nuclear export substrates or by ethidium bromide staining for tRNA (10% polyacrylamide gel). Note, eEF1A interacts with Exp5, but not with any of the other established export mediators. tRNA was retrieved specifically with both, Exp-t (ExpT) and Exp5. ‘Imp’, importin; ‘Exp’, exportin; ‘Trn’, transportin; and ‘Snp’, snurportin 1. (C) Binding of a HeLa extract to zz-tagged Exp5 in the absence or presence of 5 µM RanQ69L (GTP).

Fig. 2. eEF1A is strictly excluded from nuclei. The cellular distribution of eEF1A was studied by several methods. First, Alexa 488-labelled anti-eEF1A antibodies were used to localize the endogenous protein within cultured BHK cells. The indicated panels show the distribution of eEF1A–GFP or GFP–eEF1A fusion proteins in stably transfected BHK cell lines. Finally, GFP-tagged S2, the second isoform of eEF1A was also studied. All images represent confocal sections through the equators of the nuclei.

Fig. 3. Neither an ectopic import signal nor a block of CRM1 export is sufficient for nuclear accumulation of eEF1A. Panels show the distribution of eEF1A–GFP and eEF1A–GFP–NLS fusions in stably transfected BHK cells. The addition of the NLS only produces a faint nuclear signal that is not enhanced further by a 30 min block of CRM1 by 5 ng/ml LMB.

Fig. 4. Exp5 is a functional nuclear export receptor for eEF1A. Monospecific antibodies raised against mouse Exp5 were injected into nuclei of BHK cells expressing the eEF1A–GFP–NLS fusion. Cells were fixed 90 min post-injection. The injection marker Texas red dextran and the eEF1A–GFP–NLS fusion protein were detected in separate fluorescence channels of the confocal laser scanning microscope. Arrows indicate injected cells. Note that anti-mouse Exp5-injected cells showed strong nuclear accumulation of the eEF1A–GFP–NLS fusion. Antibodies against CRM1 or Drosophila Exp5, which do not cross-react with mammalian Exp5, had no effect on the localization of the eEF1A–GFP–NLS fusion.

Fig. 5. The interaction between Exp5 and eEF1A is mediated by tRNA. (A) A cytosolic HeLa extract was depleted of nuclear transport receptors and fractionated further to separate eEF1A from tRNA: the flowthrough from a Q-Sepharose column contained eEF1A, but lacked tRNA, while the phenol/chloroform extract contained tRNA, but no protein. Where indicated, tRNA was used after deacylation. These fractions were tested singly or in combination for binding to Exp5/RanGTP or ExpT/RanGTP as in Figure 1. The figure shows starting materials (upper parts), Exp5-bound materials (middle parts) and ExpT-bound materials (lower parts). Detection of eEF1A was by western blotting. RNA was detected after Sybr-green staining of the 12% polyacrylamide gel. The load of the bound fractions corresponds to 10 times the starting materials. Note that eEF1A binding to Exp5 was strictly RNA dependent, but tRNA binding to Exp5 occurred independently of eEF1A. Deacylation of the tRNA (which prevents the tRNA–EF1A interaction) only abolished recruitment of eEF1A into Exp5 export complexes, but still allowed efficient tRNA binding to either Exp5 or ExpT. (B) A comparison of RNA patterns when bound to Exp5 and Exp-t.

Fig. 6. The majority of translation factors show strict nuclear exclusion. Panels show confocal sections through BHK cells expressing the indicated GFP-tagged translation factors. eIF5B–GFP was introduced by transient transfection; all other translation factor–GFP fusions were expressed weakly in stable cell lines under tet control. For quantitation, see Table I; for details, see Materials and methods and main text.

Fig. 7. CRM1 excludes eIF2β from nuclei. Panels show confocal sections through stably transduced BHK cells expressing an eIF2β–GFP fusion. Where indicated, cells had been treated with 5 ng/ml LMB before fixation. Note that LMB treatment changed the eIF2β localization from completely cytoplasmic to predominantly nuclear. The duration of LMB treatment was kept short (30 min) to rule out non-specific side effects of the drug.

Fig. 8. CRM1 contributes to nuclear exclusion of several translation factors. Stably transduced BHK cells expressing the indicated translation factors were analysed. The translation factors were tagged with either GFP alone or with GFP–NLS (to increase the nuclear influx rate). The distribution of the fusion proteins was analysed by laser scanning microscopy without or after LMB treatment (5 ng/ml for 30 min).