Fig. 1. Binding of proteins from HeLa cell extract to Exp5. HeLa cell extract (lane 1) was incubated with immobilized 2zExp5 (lanes 2 and 3) or 2zExp-t (lanes 4 and 5) in the absence or presence of RanQ69L(GTP). Bound proteins were separated by SDS–PAGE and detected by Coomassie Blue staining. The arrow indicates the 50 kDa protein that was excised, subjected to mass spectrometry and identified as eEF1A.

Fig. 3. Exp5 stimulates nuclear export of eEF1A. A mixture of in vitro translated, radiolabeled eEF1A and GST was injected into the oocyte nuclei. After 2 h or at the indicated time, protein was extracted from either total oocytes (T), or, after dissection of the oocytes, from the cytoplasmic (C) and nuclear fractions (N). Proteins were separated by 10% SDS–PAGE and detected after phosphoimaging (for quantification) and autoradiography. (A) Export of eEF1A is promoted by Exp5 and requires nuclear RanGTP. Where indicated, purified 2zExp5, RanQ69L(GTP) or RanGAP (Rna1p) were co-injected. The concentrations in the injected samples were: 5 µM 2zExp5, 15 µM RanQ69L and 20 µM RanGAP. Note that only full-length eEF1A molecules were exported, while eEF1A fragments derived from the synthesis in the reticulocyte lysate stayed nuclear. (B) Nuclear export of eEF1A is not stimulated by other exportins. Co-injection of 2zExp-t (5 µM) or 2zCRM1 (5 µM) did not enhance the nuclear export of eEF1A. Both factors were fully functional, as they supported the export of GST–NES (not shown) and tRNA (Figure 6), respectively.

Fig. 5. tRNA confers binding of eEF1A to Exp5. (A) aa-tRNA stimulates binding of eEF1A to Exp5. tRNA was depleted from HeLa cell extracts by passing the extract over Q-Sepharose as described in Materials and methods. The depleted extract was supplemented with 1 mM GMPPNP to compensate for the loss of nucleotides and to allow for the efficient association of eEF1A with aa-tRNA. The starting (start) and depleted extracts (FT-Q) were subjected to binding to 2zExp5 in the presence of the factors indicated at the top of the figure. tRNA was supplemented to the FT-Q in either the aminoacylated (aa) or deacylated (da) form. 2zExp5 and bound factors were isolated from the reaction mixtures as described in Materials and methods. Then, each sample was divided into two equal parts. From one part, bound proteins were eluted and separated by 8% SDS–PAGE followed by Coomassie Blue staining or immunoblotting (WB). The load in the extract lanes (lanes 1 and 2) is 1/300 of the input, and the load from the bound fractions (lanes 3–7) corresponds to 1/12 of the total. The recruitment of eEF1A to 2zExp5 could only be detected in the presence of RanGTP and if the depleted extract had been replenished with aa-tRNA (lane 6). In comparison, the addition of tRNA that had been deacylated by treatment at high pH did not restore eEF1A binding (lane 7). Note that eEF1A migrates here as a double band of 50 and 52 kDa. Protein from both bands was subjected to mass spectrometric analysis and identified as eEF1A. (B) Exp5 binds aminoacylated and deacylated tRNA. The experiment was performed as in (A). Bound RNAs were extracted from the second part of each sample and analyzed by agarose gel electrophoresis (1.5%) and ethidium bromide staining. To demonstrate that tRNA was removed efficiently in the depleted extract, RNA was also extracted from the starting extract and the FT-Q (lanes 1 and 2). The load equals 1/30 of the total RNA retrieved from these extracts. The load in the bound fractions corresponds to 2/5 of the total. In addition, 1/15 of the aa-tRNA and da-tRNA (lanes 8 and 9) that were used to supplement the Q-Sepharose flowthrough are also shown. Note that tRNA can bind to Exp5 independently of its aminoacylation status. (C) tRNA can bind directly to Exp5. Escherichia coli tRNA (lane 1) was subjected to binding to 2zExp5 (lanes 4 and 5), 2zExp-t (lanes 6 and 7) or 2zImpβ (lanes 8 and 9) in the absence or presence of RanQ69L(GTP). 2z-tagged proteins were collected from the samples with IgG–Sepharose. The amount of unspecific binding of tRNA to IgG–Sepharose was also analyzed (lanes 2 and 3). RNA was extracted from the bound fractions, separated by 8% urea–PAGE and visualized by SYBR®Gold staining. Lane 1 shows 5% of the input RNA. The load in the bound fractions corresponds to 25% of the totally bound RNA.

Fig. 2. Cytoplasmic localization of eEF1A in HeLa cells. (A) Immunostaining of eEF1A in HeLa cells. HeLa cells were grown on coverslips to ∼30% confluency and fixed with 4% paraformaldehyde. For immunostaining, a mouse monoclonal antibody to eEF1A was used in conjunction with a fluorescein-conjugated secondary antibody. (B) Localization of EGFP–eEF1A. Detection of EGFP–eEF1A (left panel) and EGFP alone was ∼36 h after transfection and fixation of the cells with 4% paraformaldehyde. While EGFP is found in both the nucleus and the cytoplasm, EGFP–eEF1A is completely excluded from the cell nucleus. All images were taken by confocal fluorescence microscopy. Two representative images are shown for each experiment.

Fig. 4. Characterization of the eEF1A–Exp5 interaction. (A) Binding of eEF1A to Exp5 is RNase A sensitive. eEF1A was translated in vitro in the presence of [³⁵S]methionine and either mock (lanes 1, 3, 4 and 5) or RNase A treated (lanes 2, 6, 7 and 8). Binding of radiolabeled eEF1A to 2zExp5 was performed in either the absence (lanes 4 and 7) or presence (lanes 5 and 8) of RanQ69L(GTP). Then, 2zExp5 and associated factors were retrieved from the reaction mixtures with IgG–Sepharose. Control reactions for unspecific binding without 2zExp5 were performed in the presence of RanQ69L(GTP) (lanes 3 and 6). After washing, bound proteins were eluted from the beads and separated by 8% SDS–PAGE. The gel was first stained with Coomassie Blue to confirm equal retrieval of 2zExp5 and, where applicable, RanQ69L (not shown). Radiolabeled eEF1A was analyzed subsequently by phosphoimaging and autoradiography. Lanes 1 and 2 show the mock- and RNase A-treated starting lysates, respectively. The load corresponds to 15% of the total for the starting lysates and to 20% for the bound fractions. (B) tRNA is bound to Exp5 from HeLa cell extracts. HeLa cell extract was incubated with immobilized 2zExp5, 2zExp-t or 2zImpβ in the absence or presence of RanQ69L(GTP) as described in Materials and methods. Bound RNAs were separated by 8% urea–PAGE and visualized by SYBR®Gold (Molecular Probes) staining. HeLa cell tRNA is shown in the ‘input’ lane.

Fig. 6. Exp5 stimulates nuclear export of tRNA. A mixture of radio labeled tRNA_i^Met, U6Δss and constitutive transport element (CTE) RNAs was injected into the nuclei of Xenopus oocytes, either alone (lanes 1–3) or in the presence of unlabeled competitor tRNA (0.65 µg/µl in the injection mixture) derived from E.coli (lanes 4–12). Injection mixtures were supplemented with either recombinant 2zExp5 (lanes 7–9), recombinant 2zExp-t (lanes 10–12) or the corresponding buffer (lanes 1–6). Note that the concentration of 2zExp5 in the injected sample was 40 µM while that of 2zExp-t was only 10 µM. Export was analyzed 30 min after injection. T, C and N are defined as in Figure 3. Isolated RNAs were separated by 8% urea–PAGE and analyzed first by phosphoimaging for quantification and thereafter by autoradiography.