The mechanism of ribosomal export from the nucleus to the cytoplasm is not understood. Compared to the nuclear export of other cargoes, e.g., small RNAs such as tRNA, nuclear export of ribosomes is likely to be more complicated, in view of an extremely complex biogenesis pathway of ribosomes. In particular, modification, processing, and conformational rearrangement steps, which take place during ribosomal assembly, could be a trigger for transport from the nucleolus to the nuclear pores and further to the cytoplasm. Here we report the identification of a novel class of mutants, defined as rix mutants, using a visual assay for the nuclear retention of a Rpl25p-eGFP reporter. Around 2% (20 of 900) of the ts mutant strains screened gave rise to a rix phenotype, comparable to levels of mutations found for other major pathways, e.g., cell cycle progression or pre-mRNA splicing. While we do not yet know how many complementation groups these mutants represent, the first five genes tested fell into different complementation groups (data not shown). We conclude that a substantial number of genes (probably at least 50) can give rise to a rix phenotype. Analysis of these should allow the genetic dissection of ribosomal assembly and transport and possibly their coupling.
The first mutant to be analyzed, rix5-1
, was found to be due to an A68V mutation in the ribosomal protein Rpl10p. This suggests that late assembly steps during ribosome biogenesis, which occur inside the nucleus, are crucial for subsequent export to the cytoplasm. Rpl10p is conserved, having homologues in humans (28
), archea, and E. coli
). Since Rpl10p was absent from the preparation of the 66S nuclear preribosomal particle, it was proposed that Rpl10p assembles into 60S subunits in the cytoplasm (19
). However, Rpl10p is relatively weakly associated with the 60S subunit (5
) and thus may be lost from the preribosomal particles during purification.
Our findings that Rpl10p is required for nuclear pre-rRNA processing is another hint that it associates with preribosomes in the nucleus. Consistent with this interpretation, we find an NLS in Rpl10p that uses the import receptor Kap123p, which is one of two importins (Kap121p and Kap123p) involved in import of ribosomal proteins (29
). On the other hand, we do not see nuclear accumulation of Rpl10p-eGFP in nucleoporin mutants. This is not understood, but Rpl10p-eGFP, in contrast to Rpl25p-eGFP, may be more sensitive to proteolysis inside the nucleus when attached to preribosomal particles that are either not fully assembled or blocked in export. It is well known that both rRNA processing intermediates and ribosomal proteins are quickly degraded when not assembled or assembled incorrectly into preribosomes. Interesting in this context is that newly synthesized 25S rRNA is unstable (half-life of 4 min) in nmd3 ts
mutants at the restrictive temperature (13
). Apparently, 25S rRNA-containing ribosomal particles that accumulate inside the nuclei of nmd3
mutants have a short lifetime. Thus, our observation that Rpl10p-eGFP does not accumulate in the nuclei of transport mutants could reflect a high turnover rate within transport-arrested precursor particles. On the other hand, the Rpl25p-eGFP reporter appears to have a longer half-life in nuclear ribosomal precursor particles and therefore may be observed by fluorescence microscopy. Thus, it remains to be shown why a nuclear form of Rpl10p-eGFP can be detected in the rrp44/dis3-1
strain. One possibility is that inactivation of the exosome system, which functions in degradation of (aberrant) pre-rRNAs (1
), stabilizes pre-rRNA and as well ribosomal proteins bound to it.
The finding that Rpl10p is involved in a late ribosomal assembly step, which is coupled to nuclear export, together with the well-described genetic link between RPL10
) suggested that Nmd3p could play a role in large subunit export. Nmd3p has been implicated in translational control of gene expression (16
) and in formation, function, or maintenance of stable 60S subunits (3
). Strikingly, we find nuclear accumulation of the large subunit reporter Rpl25p-eGFP in several nmd3
mutants, including a slow-growing mutant lacking one of the two NESs from the C domain. When both leucine-rich NESs are deleted, the corresponding Nmd3p construct is no longer functional. Furthermore, the NES domain of Nmd3p, when attached to another NLS, exhibits a distinct NES activity. All of these findings suggest that the essential role of the Nmd3p C domain is to function in nuclear export. This is consistent with the observation that archaebacterial homologues of Nmd3p lack the NES-containing C domain characteristic of eukaryotes (3
Our data also strongly suggest that Xpo1p is the NES receptor for Nmd3p. Interestingly, an LMB-sensitive xpo1
mutant, which exhibits inhibition of nuclear export of NES-containing cargoes upon addition of LMB (27
), allows us to observe a rapid nuclear accumulation of both Nmd3p-eGFP and Rpl25p-eGFP upon inhibition of Xpo1p. In contrast, the classical xpo1-1 ts
mutant does not reveal this strong nuclear export defect of both Nmd3p and Rpl25p-eGFP. It is conceivable that the fast onset and strong inhibition of nuclear poly(A)+
RNA export in xpo1-1
) impairs ribosomal biogenesis at an early stage, because mRNAs encoding ribosomal proteins are not exported to the cytoplasm. In such a scenario, ribosomal proteins including the Rpl25p-eGFP reporter would no longer be synthesized at the restrictive temperature. What could be the reason why Nmd3p enters the nucleus and is subsequently exported to the cytoplasm by Xpo1p? A likely possibility is that Nmd3p participates in ribosomal subunit export, which correlates well with the observed concomitant nuclear accumulation of Rpl25p-eGFP and Nmd3p-eGFP in the LMB-sensitive XPO1
strain. Biochemical data further support a model in which Nmd3p binds to Rpl10p, thereby targeting Nmd3p to the 60S subunits. In the course of this work, Ho et al. characterized Nmd3p as a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit (14
), which is in full agreement with the data presented here.
Remarkably, the nuclear protein Rsa1p was implicated in facilitating the association of Rpl10p with 60S ribosomal subunits (17
). We found that like Rpl10p, Rsa1p is required for efficient nuclear export of 60S ribosomal subunits at 37°C. Interestingly, at the restrictive temperature free 60S ribosomal subunits of an rsa1
null mutant are depleted of Rpl10p (17
). The accumulation of half-mer polysomes has been reported for many mutants that underaccumulate 60S subunits, due to the presence of polysomes carrying an initiation complex that awaits a 60S subunit. This phenotype is also seen in Rpl10p mutants and strains lacking Rsa1p, but these strains show no clear change in overall 40S/60S ratios. It appears possible that this phenotype is a consequence of the retention of 60S subunits within the nucleus. Thus, Rsa1p could be a factor that mediates assembly of Rpl10p onto 60S ribosomal subunits inside the nucleus (17
In summary, we have identified several factors that function in nuclear export of 60S ribosomal subunits. We found that a protein of the 60S ribosomal subunit, Rpl10p, is required for large subunit export. This protein most likely participates in late intranuclear maturation steps leading to export-competent 60S subunits. Rpl10p interacts directly with Nmd3p, a NES-containing protein that is specifically associated with 60S subunits. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and uses the Xpo1p receptor for its export. Accordingly, the highly conserved Nmd3p could act as an adapter which requires the general NES receptor Xpo1p for export. As a consequence, 60S subunits could be exported by Xpo1p. Thus, an essential role of Xpo1p in yeast appears to be its involvement in ribosomal export. A similar conclusion that Nmd3p is an Xpo1p-dependent adapter protein for nuclear export of the large subunit has been recently published by another group (14
). Finally, it is possible that other ribosomal proteins may bind to Nmd3p or that other adapter proteins similar to Nmd3p exist. By exploiting the powerful yeast genetics, it should be possible to find additional components of the ribosomal export machinery.