Distinct biochemical and shuttling properties of the N and C termini of Xpo-t.
To investigate the biochemical properties and functional domains of Xpo-t, we generated a series of proteins with N- and C-terminal deletions and tested their ability to form an export complex containing RanGTP and tRNA in vitro (Fig. ). Protein A-tagged Xpo-t and the various deletion mutants were expressed in E. coli and purified by binding to IgG-Sepharose. The different Xpo-t proteins were then tested for RanGTP (Fig. ) and 32P-labeled tRNA (Fig. ) binding. As expected, full-length Xpo-t specifically bound to RanGTP but not RanGDP (Fig. , lanes 1 and 2) and could specifically select tRNA from a mixture of RNAs only in the presence of RanGTP (Fig. , lanes 2 and 3). Removal of C-terminal sequences eliminated the tRNA interaction (Fig. , lanes 8 to 10) but not the ability to bind RanGTP (Fig. , lanes 6 to 8). Removal of N-terminal sequences affected both binding activities (compare Fig. , lanes 4 and 5, with Fig. , lanes 6 and 7). However, removal of the first 45 amino acids of Xpo-t did not eliminate RanGTP (Fig. , lane 3) or tRNA binding (Fig. , lane 5). The tRNA interaction with Xpo-t is thus mediated by the C terminus of the protein (Fig. ), and RanGTP binds within the N-terminal 385 amino acids. Xpo-t therefore has a domain organization similar to those of other members of the importin β family.
FIG. 1. In vitro binding and shuttling activities of full-length Xpo-t and Xpo-t with N- and C-terminal truncations. (A) RanGTP binding. zzXpo-t and the truncation mutants indicated were expressed in E. coli and prebound to IgG-Sepharose, mixed with recombinant (more ...)
FIG. 2. tRNA-binding Xpo-t mutants do not affect shuttling activity. (A) Recombinant zzXpo-t mutants were tested for binding to tRNA in vitro as for Fig. . The mutants are M1 (K539A/R543A), M2 (R550A/K553A/K557A), M3 (L547A/F551A), M4 (F548A/V552A), (more ...)
Xpo-t shuttles in and out of the nucleus but is predominantly nuclear at steady state (2
). We tested the effects of Xpo-t truncations on these properties by microinjection of the 35
S-labeled proteins into Xenopus
oocytes. As previously shown (2
), when injected into either the nucleus or the cytoplasm, Xpo-t can move into the other compartment (Fig. , lanes 1 to 4) and therefore shuttles. However, when either the N or C terminus was removed, shuttling behavior was altered (Fig. and Table ). In contrast to full-length Xpo-t, N-terminal Xpo-t fragments, such as the fragment comprising amino acids 1 to 385 shown, stay in, or migrate into, the nucleus depending on where they are initially injected (Fig. , lanes 5 to 8, and data not shown). C-terminal fragments, such as amino acids 443 to 962 (see also Table ), behave in the opposite way: they are able to move to the cytoplasm (Fig. , lanes 9 and 10) but do not efficiently move into the nucleus (Fig. , lanes 11 and 12). Furthermore, glutathione S
-transferase (GST) fusions could be used to further map the export activity to the C-terminal 178 amino acids (Fig. , lanes 13 and 14). Smaller fragments did not shuttle (Fig. , lanes 17 to 20), further demonstrating the specificity of this activity, as well as indicating that the small C-terminal fragments do not leave the nucleus by simple diffusion through the NPC. Therefore, removing the N or C terminus of Xpo-t had opposite effects on the steady-state distribution of the remaining protein in Xenopus
oocytes. These properties were examined further in the experiments discussed below using Xpo-t amino acids 1 to 385 (XpN) and 443 to 962 (XpC).
In vitro and in vivo activities of Xpo-t deletion mutants
Xpo-t is predominantly nuclear because it binds to RanGTP.
One way to explain the above results is that Xpo-t contains two distinct translocation domains that impart directionality of movement to the receptor; one for import (XpN) and one for export (XpC). Alternatively, the observed steady-state localization of XpN and XpC might be due to specific interactions with nuclear or cytoplasmic components. The effect of mutations perturbing tRNA or RanGTP binding on the shuttling properties of Xpo-t were therefore tested. Since Xpo-t does not contain a sequence with similarity to known RNA binding motifs, we made the assumption that basic amino acid residues might be involved in tRNA binding. To obtain tRNA binding mutants, several highly conserved basic amino acids in the C-terminal half of the protein were mutated to alanines. Two mutants impaired in tRNA binding were identified (Fig. , lanes 5 and 8). When proteins were injected into Xenopus oocytes, no obvious differences in the steady-state distributions of these mutants relative to that of wild-type Xpo-t were observed (Fig. ). We conclude that tRNA binding does not affect Xpo-t localization.
We next tested the effect of RanGTP binding. First, 35
S-labeled Xpo-t was injected into the cytoplasm (Fig. , lanes 1 and 2) and allowed to equilibrate for 6 h (Fig. , lanes 3 and 4). Then a second injection of either RanGAP and RanBP1 (Fig. , lanes 9 to 12) or a buffer control (Fig. , lanes 5 to 8) into the nucleus was performed. Nuclear injection of GAP/BP1 depletes nuclear RanGTP pools and collapses the RanGTP gradient across the nuclear envelope (22
). After 2 to 4 h of further incubation, oocytes injected with GAP/BP1 showed dramatically reduced levels of Xpo-t in the nucleus (Fig. , lanes 9 to 12).
FIG. 3. Xpo-t is predominantly a nuclear protein at steady state because it binds to RanGTP. (A) Depletion of nuclear RanGTP alters the steady-state localization of Xpo-t. 35S-labeled Xpo-t was injected into the oocyte cytoplasm (lanes 1 and 2) and incubated (more ...)
As a second approach, we determined the steady-state distribution of a RanGTP-binding Xpo-t mutant. We generated a number of mutations in conserved residues within the Ran-binding domain and tested the RanGTP binding of the mutants in vitro. One mutant, with phenylalanines 54 and 55 changed to alanines, was defective in binding to RanGTP both in the context of full-length Xpo-t (Xpo-t mut) and within the XpN fragment (XpN mut) (Fig. , compare lanes 1 with 3 and 4 with 5). The full-length mutant was characterized by oocyte injection. Four hours after nuclear injection more mutant protein than wild-type protein was found in the cytoplasm (Fig. , compare lanes 3 and 4 with lanes 5 and 6). Additionally, when injected into the cytoplasm, very little of the mutant protein accumulated in the nucleus (Fig. , lane 11). The XpN mut protein was unstable in oocytes and could not be analyzed in this way. Taken together, these experiments suggest that nuclear RanGTP binding is required for the steady-state nuclear localization of Xpo-t.
Ran influences the levels of nuclear Xpo-t in semipermeabilized HeLa cells.
To better understand and further confirm the influence of nuclear RanGTP on Xpo-t localization, we turned to an assay of digitonin-permeabilized HeLa cells, where it is possible to more precisely control the levels of nuclear RanGTP. The ability of Xpo-t to enter the nuclei of these cells in the absence or presence of an added “Ran system” (NTF2, RanGDP, RanBP1, and RanGAP) (11
) was tested. The reactions described here and below all reflect the steady-state distribution of Xpo-t and its derivatives (data not shown). Fluorescently labeled Xpo-t enters the nucleoplasm of permeabilized cells (Fig. , control) (27
) without an energy regeneration system or Ran (data not shown). Nuclear entry of Xpo-t was blocked, as expected, by addition of a dominant-negative NPC binding fragment of importin β, ΔN44 (amino acids 45 to 465) (26
) (Fig. , +ΔN44). When the complete Ran system was included in the reaction, the amount of labeled Xpo-t in the nucleus increased (Fig. , +NRGB). The XpN protein behaved similarly to full-length Xpo-t in this assay. It entered the nucleus and was blocked by ΔN44, and nuclear accumulation was increased upon addition of a complete Ran system (Fig. ). In contrast, the XpN mut protein, deficient in RanGTP binding, entered the nucleoplasm and was blocked by ΔN44 but did not respond to the addition of a Ran system (Fig. ). These results confirm the in vivo effect of nuclear RanGTP on the accumulation of Xpo-t or XpN in the nucleus, namely, that increasing the amount of nuclear RanGTP leads to a corresponding increase in the levels of Xpo-t or XpN inside the nucleus.
FIG. 4. Localization of Xpo-t and XpN in semipermeabilized HeLa cells. The Ran system promotes nuclear accumulation. (A) Xpo-t labeled with Alexa-546 (0.5 μM) and an energy regeneration system was combined with buffer (control) or the indicated reagents (more ...) XpC localizes to the NPC and can compete for export pathways.
In permeabilized cells in the absence of an energy regeneration system or Ran, fluorescently labeled XpC accumulated at the nuclear rim and ΔN44 blocked this binding (Fig. , control and +ΔN44 and data not shown). Binding could also be competed by addition of either unlabeled XpC or unlabeled full-length Xpo-t, albeit at a higher concentration than for XpC (data not shown). Addition of the Ran system reduced the rim signal of XpC without leading to nuclear accumulation (Fig. , +NRGB). Dissection of the Ran system showed that a combination of NTF2 and Ran was sufficient to compete the rim signal (Fig. , +NR). NTF2, added alone (Fig. , +N), or a combination of Ran, RanGAP, and RanBP1 (Fig. , +RGB) did not compete. These results suggest that XpC can bind to the NPC and that this binding is competed by the NTF2/RanGDP import complex.
FIG. 5. Localization of the XpC fragment in semipermeabilized HeLa cells. Nuclear import of RanGDP competes for XpC binding to the NPC. Recombinant XpC labeled with Alexa dye was mixed with the indicated reagents and an energy regeneration system and incubated (more ...)
Because XpC has the ability to exit the nucleus (see above) and bind to the NPC, we predicted that it might compete other export pathways. To test this either BSA (Fig. , lanes 1 to 4), XpN (lanes 5 and 6), or XpC (lanes 7 and 8) was preinjected into oocyte nuclei. Next, a pre-mRNA (Ftz-pre), U1ΔSm snRNA, and a tRNA, together with U6Δss as an injection control, were injected. Relative to BSA, XpC strongly inhibited both U snRNA and tRNA export and, to a lesser extent, spliced Ftz mRNA export (Fig. , compare lanes 3 and 4 with 7 and 8). XpN inhibited all three RNA export pathways weakly (lanes 5 and 6). Quantitation of three independent experiments indicated that XpC inhibited U1 and tRNA export by 80 to 90% (Fig. ). Injection of fourfold-higher concentrations of recombinant full-length Xpo-t showed only modest inhibition of RNA export (data not shown).
FIG. 6. XpC can compete for specific export pathways. (A) Competition for RNA export. BSA (lanes 3 and 4), XpN (lanes 5 and 6), and XpC (lanes 7 and 8) were injected at 150 μM into oocyte nuclei, and oocytes were incubated for 30 min, followed by a second (more ...)
Since XpC is a fragment of Xpo-t, it is perhaps not surprising that tRNA export can be inhibited by injection of saturating amounts of this protein. However, the fact that U1 snRNA is inhibited to a similar extent is less expected. Export of U snRNAs is mediated by a complex of proteins that include export receptor CRM1/Xpo1, RanGTP, the Cap binding complex, and adapter protein PHAX (35
). The fact that XpC can inhibit U snRNA export suggests that CRM1 and Xpo-t might utilize similar NPC binding sites. We therefore tested the effects of XpC on the export of a PHAX derivative with mutations in the two major nuclear localization signals that prevent its reimport (42
) as well as other proteins exported via CRM1 such as An3, the human immunodeficiency virus type 1 Rev protein, and Snurportin1. XpN, XpC, and BSA was coinjected into nuclei of oocytes with 35
S-labeled NES proteins, and their export was assayed. As for U1 snRNA, PHAX export was strongly inhibited by XpC (Fig. , top, lanes 7 and 8) but was unaffected by XpN (Fig. , lanes 5 and 6). Interestingly, not all NES proteins were equally affected by XpC. An3 was only weakly inhibited, while both Rev and Snurportin1 were essentially unaffected (Fig. , bottom). These data suggest that XpC competes the export of some CRM1-substrate complexes but not all. It is doubtful that the differential effects are due to differences in export kinetics that reflect the affinity of each NES protein for CRM1, since Snurportin1 export is fast and Rev export is relatively slow but the export of neither is affected by XpC, while An3 and PHAX, whose export rates are intermediate, are inhibited. This suggests that different CRM1-cargo complexes require different nucleoporin interactions for their export. We also analyzed whether XpC could affect import mediated by either importin β or transportin in both oocyte and permeabilized-cell assays. We observed no inhibitory effects of XpC on import (data not shown).
Biochemical interactions between Xpo-t and nucleoporins.
The above experiments suggested that XpN and XpC might interact with different NPC components during translocation. Their biochemical interaction with nucleoporins was therefore examined. Recombinant importin β (amino acids 1 to 365, as a control), Xpo-t, XpN, and XpC were immobilized and mixed with Xenopus
egg extract in the absence or presence of RanGTP (Fig. ). A hydrolysis-deficient Ran mutant, the Q69L mutant, was used in this experiment to ensure that Ran remained bound to GTP. After incubation with extract, bound protein was eluted, separated by SDS-PAGE, and analyzed by Western blotting using monoclonal antibody MAb414 (Fig. ), anti-Xenopus
Nup153, or anti-Xenopus
CAN/Nup214 (data not shown). In this experiment the fragment of importin β comprising amino acids 1 to 365 showed clear interactions with both Nup153 and RanBP2, and the latter was RanQ69L dependent. Interestingly, the full-length importin β interaction with Nup153 was reported to be RanGTP sensitive (34
), indicating that this fragment behaves differently. Similarly, Xpo-t (lanes 6 to 8) and XpN (lanes 9 to 11) bind RanBP2 and Nup153, and both interactions required RanGTP. In the absence of added Ran, neither Xpo-t nor XpN bound detectably to any of the nucleoporins tested. In contrast to full-length Xpo-t, XpC bound CAN/Nup214 (Fig. , lanes 12 to 14), and this interaction was Ran independent. CAN/Nup214 is subject to proteolytic degradation, giving rise to two major species that were also detected by CAN-specific antibodies (data not shown). Nucleoporin p62 bound weakly to all four recombinant proteins regardless of RanQ69L addition (data not shown). No significant binding of the Xpo-t proteins to Nup98 or Nup107 was detected (data not shown).
FIG. 7. Nucleoporin binding to Xpo-t, XpN, and XpC. (A) The indicated recombinant (rec.) proteins were cross-linked to resin at high density (see Materials and Methods) and used for affinity binding. Affinity resin containing the different proteins was mixed (more ...)
Perhaps due to the above-mentioned degradation of CAN/Nup214 or to competition with other binding partners present in complete extracts (Fig. ), the XpC/CAN interaction was difficult to reproducibly detect in this assay. We therefore verified this interaction using WGA-Sepharose affinity-enriched nucleoporins (14
). WGA-binding proteins were mixed with either protein A-tagged CRM1 (together with RanGTP and BSA-NES as a positive control), Xpo-t, XpC, or XpN proteins. Unbound proteins (Fig. , lanes 2 to 5) and bound proteins (Fig. , lanes 6 to 9) were separated by SDS-PAGE and analyzed by Western blotting with MAb414 (Fig. ) or anti-CAN antibodies (data not shown). When bound to RanGTP and an NES substrate, CRM1 interacted with CAN (4
) (Fig. , compare lanes 2 and 6). XpC also efficiently bound to CAN (Fig. , compare lanes 4 and 8), and this interaction was Ran independent. In contrast, both Xpo-t and XpN interacted with CAN only weakly (Fig. , lanes 7 and 9). This result confirms that XpC can bind to CAN/Nup214 in a RanGTP-independent manner. We did not observe strong interaction of full-length Xpo-t with CAN in vitro. However, since XpC blocked the translocation cycle of Xpo-t (Fig. ) and since Xpo-t could compete the nuclear rim binding of XpC in vitro (see above), it is likely that high-affinity binding sites for XpC on the NPC also bind Xpo-t. The difference in the affinities of full-length Xpo-t and XpC for CAN may be analogous to the increased affinity for the NPC of the ΔN44 fragment of importin β compared to that of the full-length protein (26
A model for Xpo-t translocation through the NPC.
Integrating the results of the above experiments we propose the following model for some of the interactions of the tRNA export complex with the NPC during tRNA export and recycling of empty Xpo-t back into the nucleus (Fig. ; see also Discussion). Trimeric tRNA export complexes form in the nucleus, where RanGTP concentrations are high (Fig. , step 1). When bound to RanGTP, Xpo-t has a higher affinity for Nup153 (Fig. , step 2). Following translocation through the central channel, the export complex binds to RanBP2. Since sumoylated RanGAP binds to RanBP2 (29
), the likely functional consequence of this association is hydrolysis of GTP by Ran. Following hydrolysis, Xpo-t affinity for RanBP2 is reduced and it dissociates. Since XpC binds to CAN in a Ran-independent manner, we propose that full-length Xpo-t binds to CAN and is retained near the translocation channel for recycling. Furthermore, NTF2/RanGDP competes for XpC binding to the NPC and could act to reduce the receptor-NPC interaction and thus could contribute to the fraction available for NPC translocation.
FIG. 8. Model of Xpo-t translocation and recycling through the NPC. Xpo-t binds to RanGTP in the nucleus (step 1), and the export complex then binds to Nup153 (via the N terminus of Xpo-t) at the nuclear side of the NPC (step 2). The complex translocates and (more ...)
Two predictions of the model can be tested. First, if RanGTP hydrolysis is prevented, Xpo-t should accumulate at the NPC bound either to Nup153 or, more likely, to RanBP2. Second, if hydrolysis is permitted to occur, Xpo-t should also accumulate at the NPC if Ran import via NTF2 is perturbed since this will reduce competition for binding to NPC sites, including CAN. The model was tested by the permeabilized-cell assay. First, fluorescently labeled Xpo-t was imported into the nuclei. The supernatant, containing nonimported Xpo-t, was removed (approximately 80% of the total volume of the reaction mixture), and the cells were resuspended in fresh buffer minus labeled protein. A sample was removed immediately and fixed (Fig. , import 15 min). The remaining cells were aliquoted into a second set of reaction mixtures containing an energy regeneration system and either buffer, the complete Ran system, or various individual components of the Ran system and further incubated for 10 min. Note that Ran is able to cross the NPC by simple diffusion in the absence of NTF2, but less efficiently than in its presence.
FIG. 9. Xpo-t accumulates at the nuclear rim when either Ran hydrolysis or import is perturbed. (A) Two-step permeabilized-cell assay in which fluorescently labeled Xpo-t is first imported into the nuclei for 15 min at room temperature (import 15 min +…) (more ...)
Reaction mixtures containing buffer, the complete Ran system (NRGB), or NTF2 alone (N) showed a relatively diffuse Xpo-t signal within the nucleoplasm and no detectable accumulation at the nuclear rim. In contrast, omitting any component of the Ran system resulted in a reduction of the nuclear signal and some Xpo-t accumulation at the nuclear rim, i.e., preferential NPC binding. The two most relevant conditions were when NTF2 and Ran (NR) were added and when Ran, RanGAP, and RanBP1 (RGB) were present. In the first case Ran is efficiently imported and converted to RanGTP but GTP hydrolysis is inefficient due to the absence of RanGAP and RanBP1. We interpret the increase in NPC binding here as stabilization of interactions like those of Xpo-t with Nup153 and RanBP2. In the second case hydrolysis is efficient but Ran import via NTF2 is not. The net result was also a rim signal in the majority of cells, indicating that Xpo-t accumulated at the NPC. This we interpret as empty Xpo-t, after RanGTP hydrolysis, binding to the NPC through RanGTP-independent sites such as that for CAN/Nup214. The results of this experiment are consistent with some predictions of the model (Fig. ), namely, that, when a complete Ran system is present, Xpo-t can recycle rapidly between the NPC and nucleoplasm, and the net result is an evenly distributed nucleoplasmic signal and no accumulation at the NPC. However, when either GTP hydrolysis is prevented or the NTF2/RanGDP complex is absent, Xpo-t accumulates at the NPC. The overall reduction in fluorescence signal in these experiments reflects the Xpo-t that is exported completely, escaping binding to the NPC and reimport, and that is diluted into the buffer phase.