Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.     

Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.     

Novel interaction of the 60S ribosomal subunit export adapter Nmd3 at the nuclear pore complex

Nuclear export of the large (60S) ribosomal subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.     

A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains. In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Chemical crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre-60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNA and ribosome export pathways.     

Mapping the functional domains of yeast NMD3, the nuclear export adapter for the 60 S ribosomal subunit

Nuclear export of the large ribosomal subunit requires the adapter protein Nmd3p to provide a leucine-rich nuclear export signal that is recognized by the export receptor Crm1. Nmd3p binds to the pre-60 S subunit in the nucleus. After export to the cytoplasm, the release of Nmd3p depends on the ribosomal protein Rpl10p and the GTPase Lsg1p. Here, we have carried out a mutational analysis of Nmd3 to better define the domains responsible for nucleocytoplasmic shuttling and ribosome binding. We show that mutations in two regions of Nmd3p affect 60 S binding, suggesting that its binding to the subunit is multivalent.     

Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.     

The mRNA export factor Npl3 mediates the nuclear export of large ribosomal subunits

The nuclear export of large ribonucleoparticles is complex and requires specific transport factors. Messenger RNAs are exported through the RNA-binding protein Npl3 and the interacting export receptor Mex67. Export of large ribosomal subunits also requires Mex67; however, in this case, Mex67 binds directly to the 5S ribosomal RNA (rRNA) and does not require the Npl3 adaptor. Here, we have discovered a new function of Npl3 in mediating the export of pre-60S ribosomal subunit independently of Mex67. Npl3 interacts with the 25S rRNA, ribosomal and ribosome-associated proteins, as well as with the nuclear pore complex. Mutations in NPL3 lead to export defects of the large subunit and genetic interactions with other pre-60S export factors.     

Ltv1 is required for efficient nuclear export of the ribosomal small subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Deltaltv1 mutants, as judged by the altered distribution of the 5'-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.     

Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.     

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.     

Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.     

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.     

Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p

In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.     

Structural similarity in the absence of sequence homology of the messenger RNA export factors Mtr2 and p15

The association between Mtr2 and Mex67 is essential for the nuclear export of bulk messenger RNA in yeast. In metazoans, the analogous function is carried out by the TAP–p15 heterodimer. Whereas Mex67 and TAP are highly conserved proteins, their binding partners, Mtr2 and p15, share no sequence similarity, but are nevertheless functionally homologous. Here, we report the 2.8-Å resolution crystal structure of Mtr2 in complex with the NTF2-like domain of Mex67. Mtr2 is a novel member of the NTF2-like family and interacts with Mex67, forming a complex with a similar structural architecture to that of TAP–p15. Mtr2 fulfils an analogous function to that of human p15 in maintaining the structural integrity of the heterodimer. In addition, Mtr2 presents a long internal loop, which contains residues that affect the export of the large ribosomal subunit.     

The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm,respectively

We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p. Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus. Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature. Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus. Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect. We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.     

Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

Assembly of the Trypanosoma brucei 60S ribosomal subunit nuclear export complex requires trypanosome-specific proteins P34 and P37

We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.     

Mutational analysis of the ribosomal protein Rpl10 from yeast

Yeast Rpl10 belongs to the L10e family of ribosomal proteins. In the large (60 S) subunit, Rpl10 is positioned in a cleft between the central protuberance and the GTPase-activating center. It is loaded into the 60 S subunit at a late step in maturation. We have shown previously that Rpl10 is required for the release of the Crm1-dependent nuclear export adapter Nmd3, an event that also requires the cytoplasmic GTPase Lsg1. Here we have carried out an extensive mutational analysis of Rpl10 to identify mutations that would allow us to map activities to distinct domains of the protein to begin to understand the molecular interaction between Rpl10 and Nmd3. We found that mutations in a central loop (amino acids 102-112) had a significant impact on the release of Nmd3. This loop is unstructured in the crystal and solution structures of prokaryotic Rpl10 orthologs. Thus, the loop is not necessary for stable interaction of Rpl10 with the ribosome, suggesting that it plays a dynamic role in ribosome function or regulating the association of other factors. We also found that mutant Rpl10 proteins were engineered to be unable to bind to the ribosome accumulated in the nucleus. This was unexpected and may suggest a nuclear role for Rpl10.