1. Identification of a tRNA-Specific Nuclear Export Receptor
Ulrike Kutay, Gerd Lipowsky, Elisa Izaurralde, F.Ralf Bischoff, Petra Schwarzmaier, Enno Hartmann, Dirk Görlich 
Molecular Cell 
Volume 1, Issue 3, Pages (February 1998)
DOI: /S (00)

2. Figure 1
Identification of RanGTP-Binding Proteins as Potential Mediators of Nucleocytoplasmic Export
A HeLa cell extract was subjected to binding to either immobilized Ran wild type (mimicking RanGDP) or to immobilized RanQ69L (mimicking RanGTP). Starting material and bound fractions were analyzed by SDS–PAGE (8% gel) followed by Coomassie staining. Peptide sequencing identified the established RanGTP-binding proteins importin β, transportin, CAS, and RanBP7 in the RanGTP-bound fraction. In addition, we found two peptides (FSSTLxNxQT, LQEFIPLINQITAK) corresponding to a novel protein, referred to here as exportin-t. Western blotting confirmed that exportin-t has been enriched on the RanGTP column to a similar extent as importin β.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2 Molecular Cloning of Exportin-t
Exportin-t was cloned from HeLa cell cDNA with the aid of the partial peptide sequence information (see Figure 1). For details, see Experimental Procedures. This figure shows the amino acid sequence of exportin-t aligned to Los1p, which is the most similar protein from S. cerevisiae. The sequence identity is 19%. (#) indicates the region against which the anti-exportin-t antibody was raised. The peptides obtained by protein sequencing are indicated.
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3 Dynamic Cellular Localization of Exportin-t
(A) Steady-state distribution of exportin-t in HeLa cells. Cells were grown on coverslips, fixed for 10 min with 2% paraformaldehyde, permeabilized with 0.1% Triton X % SDS, and blocked in 2% BSA + 10% donkey normal serum. The sample was then incubated with the affinity-purified anti-exportin-t antibody (rabbit) followed by detection with a fluorescein-conjugated secondary antibody (donkey anti-rabbit). The fluorescence signal was recorded with a confocal laser scanning microscope.
(B) Effect of Ran on exportin-t localization. Exportin-t was expressed in E. coli, purified, and modified at a 1:1 molar ratio with fluorescein 5′maleimide. The labeled protein (1 μM) was incubated with permeabilized HeLa cells in the absence or the presence of 3 μM RanQ69L. An energy-regenerating system was present in both incubations. After 10 min, the nuclei were fixed, spun onto coverslips, and analyzed by confocal laser scanning microscopy. Note that without Ran, exportin-t strongly accumulated in the nucleoplasm, whereas it was mainly detected at the NPCs when RanQ69L was present.
(C) The exit of exportin-t from the nucleus is Ran-dependent. 0.8 μM fluorescent exportin-t was allowed to accumulate for 12 min in nuclei of permeabilized cells (without energy). After 12 min, the sample was split into three, and an energy-regenerating system and either buffer, 5 μM Ran wild type, or 5 μM of the GTPase-deficient RanQ69L mutant were added. Samples were fixed 30 min later and analyzed as in (B). Note that addition of wild-type or mutant Ran changed the localization of exportin-t from nucleoplasmic to mainly NPC-associated.
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4 Exportin-t Binds tRNA
(A) Exportin-t mediates tRNA binding to RanGTP. 1.5 ml of HeLa cytoplasmic extract alone, or supplemented with either 0.7 μM exportin-t, CAS, or transportin, was subjected to binding to immobilized RanQ69LGTP (2.5 nmol). The starting material and the bound fractions were each split into two and either analyzed by SDS–PAGE followed by Coomassie staining as in Figure 1 (1/20 of the bound fraction) or extracted with phenol/chloroform and analyzed by denaturing gel electrophoresis and ethidium bromide staining (load was 1/3 of the bound fraction). Note that addition of exogenous exportin-t resulted in a significant recovery of tRNA in the RanGTP-bound fraction.
(B) Exportin-t binds tRNA in a RanGTP-dependent manner. Indicated combinations of 1.5 ml cytoplasmic HeLa extract or an RNA fraction from the HeLa extract (∼1100 pmol of tRNA), and 1.6 μM RanQ69L were bound to either immobilized exportin-t or transportin (150 pmol of each). Bound fractions were extracted with phenol/chloroform and analyzed by denaturing gel electrophoresis and ethidium bromide staining (load was 1/25 of the starting material and 1/3 of the bound fractions). Note that tRNA bound to exportin-t from the HeLa extract and from the RNA fraction. Binding was enhanced by RanQ69L. The stoichiometry of tRNA bound to exportin-t in the presence of the Ran mutant was close to 1:1. Immobilized transportin had been included as a negative control and did not bind RNA detectably.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5
Kinetic Characterization of the Exportin-t/tRNA/RanGTP Interaction
(A) GTPase activation on Ran is prevented in the trimeric tRNA/exportin-t/RanGTP complex. 50 pM Ran-[γ-32P]GTP was incubated for 15 min with indicated final concentrations of exportin-t in the absence or presence of 1 μM in vitro transcribed tRNASer or 1 μM importin α. 20 nM RanGAP1 (Rna1p from S. pombe) was added, and the reaction was allowed to proceed for 2 min. Hydrolysis of Ran-bound GTP was determined as released [32P] phosphate using the charcoal method. The dose dependence of GTPase inhibition can be used to estimate the constants for dissociation of RanGTP from the complexes, which is roughly 3 nM for the trimeric tRNA/exportin-t/RanGTP complex. Note that Ran and tRNA binding to exportin-t is highly cooperative, with tRNA apparently increasing exportin-t's affinity for RanGTP 300-fold.
(B) Measurements were performed exactly as in (A), except that CAS instead of exportin-t was added. Note that tRNA binds selectively to exportin-t, but not to CAS. In turn, importin α binds specifically to CAS but not to exportin-t.
(C) Exportin-t distinguishes tRNA from other RNAs. 50 pM RanGTP was incubated for 15 min with a mixture of 40 nM exportin-t and the various in vitro transcribed RNAs at indicated final concentrations. The formation of the tRNA/exportin-t/RanGTP complex was measured as the decrease in GAP sensitivity of RanGTP as described in (A). Note that the various tRNAs gave superimposable concentration curves and thus bind exportin-t with comparable affinity. In contrast, the binding of U1ΔSm RNA or U6 ss RNA was at least 100 times weaker.
(D) Experiment was performed as in (C), measuring the binding of the following RNAs: RRE (in vitro transcribed Rev response element); in vitro transcribed tRNASer; tRNASerΔCCA (tRNASer lacking the 3′CCA end); HeLa tRNA (tRNA isolated from Hela cells as described in the Experimental Procedures); and calf liver tRNA (Boehringer).
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 6
Disassembly of the tRNA/Exportin-t/RanGTP Complex by RanBP1+RanGAP
(A) 50 pM Ran-[γ-32P]GTP was incubated with 200 nM exportin-t and 400 nM tRNASer in a volume of 300 μl. After 15 min, 20 nM RanGAP was added, followed by the addition of either buffer or 25 nM RanBP1. GTP hydrolysis was determined in 50 μl aliquots at the indicated time points. Note that in the absence of RanBP1, the tRNA/exportin-t/RanGTP complex is entirely GAP-resistant and has a half-life of roughly 40 min. Nearly instantaneous GTP hydrolysis occured in the presence of RanBP1.
(B) Concentration dependence of the RanBP1 effect. 100 nM trimeric complex was preformed by a 15 min preincubation of 100 nM RanGTP with 400 nM exportin-t and 800 nM tRNASer. Then, 20 nM RanGAP1 was added, followed 1 min later by the addition of RanBP1 to the final concentrations indicated. GTP hydrolysis was allowed to proceed for 2 min and determined as in (A). Note that the half-maximal effect of RanBP1 was observed at a concentration of 1 nM. At this concentration, each RanBP1 molecule had catalyzed on average the disassembly of 50 tRNA/exportin-t/ RanGTP complexes.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7 Exportin-t Stimulates Nuclear Export of tRNA
(A) Effect on tRNALeu export. Xenopus laevis oocyte nuclei were coinjected with a mixture of 32P-labeled RNAs and buffer, or 5 μM recombinant exportin-t or CAS. The mixture of RNAs consisted of: DHFR mRNA, U1ΔSm, U6Δss, and tRNALeu. U6Δss does not leave the nucleus and is an internal control for nuclear integrity. RNA samples from total oocytes (T) or cytoplasmic (C) and nuclear (N) fractions were collected either immediately after injection in lanes 1–3, or after 30 min (lanes 4–12). RNAs were resolved on 8% acrylamide–7 M urea denaturing gels. Note that coinjection of exportin-t selectively accelerated export of tRNALeu (lanes 7–9), whereas coinjection of CAS had no effect (lanes 10–12).
(B) Exportin-t stimulates export of tRNAiMet and tRNASer. The experiment was essentially performed as in (A). The mixture of RNAs injected consisted of DHFR mRNA, U1ΔSm, U6Δss, tRNAiMet, and tRNASer. Export of these two tRNAs is very fast and essentially complete after 30 min (lanes 4–6). Coinjection of 4 pmol of unlabeled tRNASer competitor per oocyte significantly reduced export of tRNAiMet and tRNASer (lanes 7–9). Coinjection of 5 μM exportin-t could overcome the retardation and restimulated tRNA export to the original level (lanes 10–12).
Molecular Cell 1998 1, DOI: ( /S (00) )