3.2 Nuclear transport factors might act as compartment-specific inhibitors of
3.2.2 Addition of Crm1 to an in vitro translation assay decreases transla-
Due to the wide cargo range of Crm1 and the finding that this nuclear export factor can pull out almost all translation (initiation) factors from a HeLa cell lysate in a Ran de-pendent manner (unpublished data by K. Kirli), we wanted to analyze the effect upon Crm1 addition to an in vitro translation assay. In comparison to Exportin 4, that only interacts with specific cargo proteins and thus the reporter constructs needed to fulfill certain requirements, translation rates of a variety of different reporters should be affected upon Crm1 addition. Here, the poly-serine, poly-proline and Firefly luciferase reporters were used.
When adding recombinantly expressed and purified Crm1 WT, the reporter constructs are translated slightly less efficient as compared to a non-supplemented extract. However, by additionally supplementing the extract with Ran (in 3-fold excess over Crm1), the
trans-3.2 Nuclear transport factors might act as compartment-specific inhibitors of translation 88
lation efficiency drops significantly so that no reporter signal can be observed any longer.
The same holds true when adding Crm1G502A, a Ran independent Crm1 variant. The ef-fect seems to be concentration dependent, as it can only be observed when adding>3µM Crm1. When supplementing the in vitro translation assay with other nuclear transport factors such as Exportin 6, mediating actin export (St¨uvenet al., 2003) or CAS, respon-sible for recycling Importin α to the cytoplasm (Kutay et al., 1997), the effect can not be observed, neither in absence nor presence of Ran (see figure 2.12). These observations argue that the decrease in translation is related to the addition of nuclear transport factors indeed being known to interact with translation factors.
However, it is unlikely to assume, that Crm1 is able to bind each single subunit directly.
When analyzing the translation factors pulled out from HeLa cell lysate by Crm1, only a minor portion harbors a sequence stretch that has the properties to act as a nuclear export signal (NES). Interestingly, each multisubunit complex contains at least one subunit with a predicted NES, which seems to be enough for co-purification of the entire complex in the pull-out assay. But what happens in the in vitro translation assay?
If one assumes that certain translation factors or translation factor subunits bind to Crm1 via their NES, they most likely are sequestered because important interaction sites are shielded by Crm1. Another explanation might be that even if a functional site of a trans-lation factor would still be exposed, the Crm1:transtrans-lation factor complex could simply be too large to act during translation (initiation). Crm1 might also promote a platforming effect, meaning that once a translation factor is bound, further interacting factors assemble on to it. Thereby more and more translation factors are taken out of the actual transla-tion process and translatransla-tion efficiency drops. The observed slight decrease when adding Crm1 only might be explained by some non-Ran mediated binding of translation factors to Crm1.
So far, we hypothesize that binding and thus sequestration of the translation factors to Crm1 happens by NES binding to the hydrophobic cleft of Crm1. Hence, blocking or mu-tating the hydrophobic cleft should be able to reverse the decreased translation efficiency.
The hydrophobic cleft was blocked by either supplementing the extract with NS2 peptide, which shows high affinity towards Crm1s hydrophobic cleft (Engelsma et al., 2008), or Snurportin1 fused to either PKI-NES or REV-NES to further strengthen the interaction between Crm1 and its natural cargo. In addition, the translation assays were performed with mutant versions of Crm1, which at least in vitro are not able to bind a PKI peptide any longer (see figure 2.13). Surprisingly, the observed decrease of translation efficiency upon Crm1 and Ran addition could not be reversed by any of the applied approaches.
There are a variety of possible explanations why the observed effect could not be reversed so far. Although it is known that the NS2 peptide can bind Crm1 strongly in anin vitro
3.2 Nuclear transport factors might act as compartment-specific inhibitors of translation 89
binding assay, the conditions in a lysate are very different. Not only that the peptide could easily be degraded by proteases in the extract, there might in addition be structures in the lysate to which the NS2 peptide is attracted. Thus, we can not ensure that the NS2 peptide reaches and binds Crm1, thereby blocking the hydrophobic cleft or competing out translation factors. Even if it does reach Crm1, the affinity of the NS2 peptide towards Crm1 might not be strong enough to displace pre-bound cargos.
Next, the PKI-Snurportin or REV-Snurportin proteins were added with the aim to block the hydrophobic cleft of Crm1 in the extract. The first Crm1 crystal structure was ob-tained in complex with Ran and Snurportin1, a natural Crm1 cargo described in 1999 (Paraskeva et al., 1999; M¨onecke et al., 2009). Interestingly, the interaction between Crm1 and Snurportin1 is not exclusively via the hydrophobic cleft but Snurportin1 also interacts with several other residues on Crm1s HEAT-repeats 12-14, thereby shielding a considerable portion of Crm1s surface, making it inaccessible for other cargoes. Snur-portin1 was used as fusion protein to either PKI-NES or PEV-NES to further increase the binding affinities. Surprisingly, also supplementing thein vitro translation assay with these proteins did not lead to a rescue of translation efficiency. However, Crm1 was not pre-complexed with the Snurportin1 fusion proteins before addition to the extract. Thus, the problems of accessibility and competition for binding in the lysate still remains. As already described, addition of Crm1 hydrophobic cleft mutants (Crm1A451R, Crm1K568E) which can no longer interact with PKI-NES peptidesin vitro (unpublished data, H. Chug), also led to a significant decrease in translation efficiency of the reporter constructs. Yet another approach to block Crm1 towards NES binding is to use the toxin leptomycin B that specifically binds to the hydrophobic cleft, thereby covalently modifying a cysteine residue (Nishiet al., 1994; Kudoet al., 1998). However, leptomycin B can not be solube-lized to high concentrations needed to efficiently block Crm1 in the lysate.
When looking at Exportin 4, the decrease in translation efficiency is - as expected - only observed on poly-proline containing proteins, which once again claims that eIF5A, the cargo of Exportin 4 plays a role in the translation of poly-proline proteins. In case of Crm1 however, a broad range of cargoes can be recognized and the binding mode of nu-clear export factor and cargo is different to the binding mode of other NTRs. Most nunu-clear export factors bind their cargos by wrapping around them; the cargo interacts mainly with the inner surface of the transport receptor. Crm1 however, has in comparison a rather small interacting surface with the cargo which is also located more at the outer side of the HEAT repeats. The interaction between the NES and the hydrophobic cleft of Crm1 is based on a short, hydrophobic amino acid stretch. Also more and more non-classical NES motives are identified. Taking together, Crm1 seems to have less stringent modes of binding towards its cargo, thus the binding and sequestration of Crm1 towards the
3.2 Nuclear transport factors might act as compartment-specific inhibitors of translation 90
translation factors must not necessarily happen via the hydrophobic cleft only, explaining why even in presence of hydrophobic cleft mutants, Crm1 seems to be able to efficiently block translation. One can nevertheless also argue that the effect might be unspecific, caused by a folding stress mechanism acting on translation and triggered upon addition of possibly misfolded eukaryotic proteins expressed in E.coli. This would explain, why the effect can not be reversed by specifically blocking the interaction between Crm1 and its cargos. Other findings, namely the decrease of translation rates in a Ran dependent manner and the possibility to add other nuclear transport factors expressed in E.coli to the extract without affecting translation rates, however do suggest that Crm1 (or Exportin 4) are specifically provoking the observed translation efficiency decrease.