Our delineation of an Nmd3p- and Crm1p-dependent export pathway for the 60S ribosomal subunit is the first report of a nuclear export pathway for ribosomal subunits. Because the NES of Nmd3p is essential for 60S subunit biogenesis and export, Nmd3p appears to be the principal protein providing the export signal for the large ribosomal subunit. Although Nmd3p may have an additional role on the 60S subunit (see below), the ability to modulate 60S subunit export by the presence or absence of an NES on Nmd3p clearly demonstrates that one essential function of Nmd3p is to provide the NES for 60S export. Thus, Nmd3p acts as an adapter protein to bridge the interaction between the 60S subunit and its export receptors. Furthermore, the demonstration that Crm1p is a receptor for Nmd3p to mediate 60S subunit transport is the first evidence that Crm1p is involved in ribosomal subunit export.
Nmd3p is a highly conserved protein. Similar proteins are found throughout eukaryotes and all of the eukaryotic proteins show a high degree of conservation of the shuttling signals that we have identified within Nmd3p. We have recently cloned the human homologue, CGI-07, and found that it complements a temperature-sensitive nmd3 mutant, suggesting conservation of function of Nmd3p throughout eukaryotes (Johnson, A., unpublished results). Interestingly, related proteins are predicted in archaebacteria as well. However, these archaebacterial proteins lack the shuttling sequences that we have defined in Nmd3p. Thus, eukaryotic Nmd3 proteins appear to have evolved by the addition of an NLS and NES, thereby adapting an archaea-like protein for nuclear shuttling.
Since the COOH-terminal 50 aa of Nmd3p (aa 469–518) is necessary for nuclear export, this domain of the protein likely contains an NES. Within this region, aa 491–500 (INIDELLDEL) are highly conserved and are predicted to form an amphipathic helix with isoleucine and leucine predominantly on one face, similar to a leucine-rich NES (Rittinger et al. 1999
). Because export of Nmd3p depends on Crm1p, the receptor for leucine-rich NES–containing proteins, we tentatively conclude that aa 491–500 comprise the NES of Nmd3p. Experiments to determine the minimal NES of Nmd3p are underway. We note that Nmd3Δ100 displayed a stronger dominant-negative phenotype than Nmd3Δ50. The larger deletion in Nmd3Δ100 encompassed aa 419–468, which contains an additional highly conserved domain. Preliminary results indicate that this region is not necessary for function, but may act additively with aa 469–518 (Johnson, A., unpublished results). This region could encode a second, but weaker NES, or a signal for intranuclear localization. Consequently, it is possible that there is redundancy in the export signal and possibly in the export pathway. Determination of the intranuclear localization of mutant Nmd3 proteins deleted for these various signals should elucidate their respective contribution to Nmd3p localization.
Leptomycin B is an antibiotic specific for Crm1p in nearly all eukaryotic cells (Nishi et al. 1994
; Kudo et al. 1999
). Wild-type S. cerevisiae
cells are resistant to leptomycin B. However, a single aa change within Crm1p renders S. cerevisiae
sensitive to the antibiotic (Neville and Rosbash 1999
). Since Nmd3p export was inhibited by leptomycin B, we conclude that Crm1p is the receptor for Nmd3p. In preliminary in vitro experiments, we have also observed Ran binding in the presence of both Crm1p and Nmd3p (Kallstrom, G., and A. Johnson, unpublished results), which suggests the cooperative interaction of Ran and Crm1p in the formation of an export complex (Fornerod et al. 1997
; Kutay et al. 1998
). Furthermore, we showed that Crm1p is needed for efficient 60S subunit export. This is contrary to a previous report in which temperature-sensitive crm1(xpo1-1)
mutant cells did not inhibit 60S subunit export when shifted to restrictive temperature (Hurt et al. 1999
). We also found that Nmd3p did not accumulate in the nucleus in crm1(xpo1-1)
cells at restrictive temperature. Preliminary results suggest that this failure to observe Nmd3p accumulation in the nucleus was not due to the inhibition of Nmd3p import at restrictive temperature (Ho, J., and A. Johnson, unpublished results). The transient inhibition of ribosome biogenesis due to temperature shifts (Warner 1999
) likely complicates the use of crm1(xpo1-1)
mutants for examining effects on ribosome export. It is also possible that an alternative and Crm1p-independent pathway acts at elevated temperature to bypass the Crm1p-dependent pathway. Nevertheless, we did observe a strong nuclear accumulation of Nmd3p and 60S subunits in leptomycin B–sensitive cells when treated with leptomycin B. Thus, Crm1p is an export receptor for Nmd3p to mediate 60S subunit export.
The shuttling of Nmd3p in and out of the nucleus depends on the recognition of import and export signals by receptor proteins. When Nmd3p binds to 60S subunits in the nucleus, its NES must be displayed for recognition by Crm1p. Nmd3p also binds mature 60S subunits in the cytoplasm (Ho and Johnson 1999
; Ho et al. 2000
). Consequently, the NLS of Nmd3p bound to 60S subunits in the cytoplasm must be masked to prevent retrograde transport of mature subunits to the nucleus. A similar proposal has been made for ribosomal proteins (Rout et al. 1997
). Because Nmd3p is predominantly cytoplasmic, where it binds mature free 60S subunits (Ho et al. 2000
), the ratio of Nmd3p to free 60S subunits in the cytoplasm may determine the availability of Nmd3p for shuttling into the nucleus.
Does Inhibition of 60S Subunit Export Affect Other Transport Pathways?
In a screen for high-copy suppressors of the growth defect of an nmd3-1
mutant (Ho and Johnson 1999
) we identified MEX67
, encoding an mRNA transport factor (Segref et al. 1997
; Hurt et al. 2000
), and PAB1
, encoding poly(A) binding protein (Kallstrom, G., and A. Johnson, unpublished results). In addition, mex67-5
mutations were synthetic lethal (Kallstrom, G., and A. Johnson, unpublished results), however, NMD3
was not a high-copy suppressor of mex67-5
. Although high-copy MEX67
partially suppressed the growth defect of nmd3-1
cells, they did not reverse the 60S subunit deficit. Consequently, MEX67
are unlikely to be directly involved in 60S export. It is possible that inhibition of 60S export indirectly affects export of mRNA leading to a condition in which mRNA is partially limiting in cells. A link between mRNA transport and the nucleolus, the site of ribosome biogenesis, has been suggested previously (Schneiter et al. 1995
). It is possible that overexpression of MEX67
partially bypasses this block, whereas overexpression of PAB1
may stabilize mRNAs (Caponigro and Parker 1995
) under conditions in which mRNA is limiting in the cell. Further work is needed to determine the basis of these genetic interactions.
Is There a More Fundamental Function for Nmd3p?
Eukaryotes appear to have adapted an archaeal Nmd3p-like protein for transporting the 60S subunit across the nuclear envelope. The presence of Nmd3p-like proteins in archaebacteria, which lack nuclei, suggests that Nmd3p has an additional function more ancient than nuclear export. Such a role could be in a biogenesis step of the large ribosomal subunit that is distinct from, but perhaps coupled to, export of the subunit. The requirement of Nmd3p for an ultimate maturation step, before export, could provide a mechanism of control of 60S export. Therefore, Nmd3p could provide a quality control mechanism for ribosomal subunit biogenesis analogous to the role of nuclear aminoacylation of tRNAs required for tRNA export (Lund and Dahlberg 1998
). We note that truncated Nmd3p, lacking an export signal, binds to nuclear 60S subunits, which are sufficiently stable to accumulate in the nucleus. However, a temperature-sensitive nmd3
mutant does not allow such nuclear accumulation of nascent 60S subunits due to their severe instability. Thus, Nmd3p also provides a function in subunit biogenesis that is necessary for stabilization of the nascent 60S subunit. We suggest that eukaryotes have adapted a ribosome biogenesis factor for transport of the large ribosomal subunit.