Here we have shown that the shuttling factor Arx1 acts as a nuclear export receptor for the 60S ribosomal subunit. This conclusion is based on several different lines of evidence. ARX1
shows genetic interaction with multiple genes encoding factors involved in 60S subunit export. These include the 60S export adapter Nmd3 and its receptor Crm1, the mRNA export receptors, Mex67 and Mtr2, which have recently been shown to be required 60S export as well, and various nucleoporins. We found that in the absence of Arx1, export of pre-60S particles was impaired, leading to accumulation of Nmd3-bound pre-60S particles in the nucleus. Surprisingly, the Nmd3-bound subunits also contained elevated levels of Crm1 as well as Mex67 and Mtr2. The nuclear accumulation of pre-60S particles loaded with export receptors suggests that Arx1 functions downstream or in concert with these receptors to facilitate export of the 60S subunit. Finally, we showed that Arx1 interacts with a subset of nucleoporins by two-hybrid assay and by in vitro binding, consistent with a previous report identifying Arx1 among proteins that could be affinity purified from yeast extracts using nucleoporins for bait (Allen et al., 2001
). Taken together, these results imply that Arx1 is a third nuclear export receptor for the 60S subunit. Similar work showing that Arx1 acts as an export receptor has recently been reported (Bradatsch et al., 2007
Sequence comparisons indicate that Arx1 has evolved from the family of type II methionyl aminopeptidases (MetAPs; Hung and Johnson, 2006
). However, catalytic residues in the active site are not conserved in Arx1, suggesting that it is not an active peptidase. Indeed, the human ortholog of Arx1, Ebp1, does not have peptidase activity (Monie et al., 2007
). These proteins have also diverged from MetAPs by the inclusion of loops and a C-terminal extension that, in Ebp1, provides an RNA-binding domain (Monie et al., 2007
). It remains to be determined if human Ebp1, which is involved in pre-60S metabolism in human cells (Squatrito et al., 2004
), also plays a role similar to that of Arx1 as an export receptor as the loops in Arx1 are much more extensive than those in Ebp1 and could provide additional interaction surfaces not found in Ebp1. This may be reminiscent of the acquisition of ribosome binding activity by Mex67 and Mtr2, a function specific to protein loops unique to the yeast proteins and not found in the metazoan proteins (Yao et al., 2007
). On the other hand, nucleophosmin has been reported to be required for export of 5S rRNA, and by extension, the 60S subunit, in human cells (Yu et al., 2006
). Nucleophosmin does not have an obvious ortholog in yeast. Thus, among the export adaptors for the large subunit, only Nmd3 appears to be well conserved in function as an export adapter. Nmd3 may represent the primordial 60S export factor for eukaryotic cells and additional adapters may have evolved independently in different eukaryotic lineages.
The inner channel of the nuclear pore complex is largely composed of FG-repeat–containing nucleoporins that create a hydrophobic meshwork, posing a permeability barrier that selectively controls the translocation of macromolecules (Ribbeck and Gorlich, 2001
; Denning et al., 2003
). Translocation of hydrophilic cargo through this hydrophobic channel of the NPC requires a mechanism for partitioning the cargo into such an environment. It has been speculated that large cargo molecules require multiple receptors, and this has been demonstrated for protein import in HeLa cells (Ribbeck and Gorlich, 2002
). On the other hand, the addition of a second import receptor synergistically stimulated import. The large subunit of the ribosome may be the bulkiest cargo to pass through the NPC. In addition, it is highly electronegative, due to the large amount of RNA on the surface of the subunit. Our results here with Arx1, combined with the previous demonstration that Nmd3 (Ho et al., 2000b
; Gadal et al., 2001
) and more recently the Mex67/Mtr2 heterodimer (Yao et al., 2007
) are required for export suggest that at least three receptors are used for efficient export in yeast. Considering the impact of nmd3
mutants on 60S export, and that Arx1 appears to be a stoichiometric component of the pre-60S complex, it seems likely that these receptors are present simultaneously on the subunit and are required in concert, rather than as alternative export pathways. We might expect these receptors to be distributed over the surface of the large subunit to allow the entire surface of the ribosome to partition into the NPC. Preliminary results suggest that Arx1 binds in the vicinity of the exit tunnel (Hung and Johnson, 2006
) whereas Nmd3 appears to bind to the joining surface (Sengupta, Bussiere, Frank and Johnson, unpublished data) on the opposite face of the subunit. The Mex67/Mtr2 heterodimer is suggested to bind 5S rRNA (Yao et al., 2007
), again potentially distal to Arx1 and Nmd3. In the case of nuclear import of large cargo molecules in HeLa cells, single import receptors were not sufficient for efficient translocation, but did promote tethering of cargo at the NPC. We have not observed tethering of preribosomal complexes at the NPC under conditions in which export is inhibited.
If multiple receptors are required for export, why are multiple different receptors used rather than multiple copies of a single receptor species? One possibility is that export with multiple different receptors is more efficient than multiple receptors of the same protein species, possibly avoiding competition between receptors for common binding sites. In addition, the different affinities of different receptors for their binding sites on nucleoporins could help to orient the ribosomal subunit with respect to the central channel of the NPC to facilitate its entry.
The presence of multiple receptors on the large subunit also raises the question of how subunit export is regulated. We have proposed previously that Nmd3 may act as a structural proofreading factor whose binding would depend on the proper assembly of a complex binding site that is presented only upon proper maturation of the subunit (Johnson et al., 2002
). Thus, the loading of Nmd3 would dictate the time of export. However, with multiple receptors, this seems a more complicated proposition. Would their loading be coordinated, or is there a hierarchy in their function, i.e., would one factor, such as Nmd3, be the primary determinant for release from the nucleolus or docking at the NPC whereas the other factors load later or perhaps function only at the NPC? Our preliminary results suggest that there is not a rigid hierarchy to the loading of these proteins. However, the accumulation of particles in the nucleus when one export receptor is disrupted could result in accumulation of Crm1 and Mex67/Mtr2 on these particles, possibly during abortive cycles of docking and release from the NPC.