Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export.

Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.

In eukaryotic cells, protein synthesis requires the synthesis, processing, and export of mRNA. mRNA export occurs through nuclear pore complexes (NPCs), 1 very large macromolecular assemblies (60 MDa in yeast and about 120 MDa in higher organisms) that are the only channels for transport of molecules and macromolecules between the nucleus and the cytoplasm. NPCs are composed of multiple copies of ϳ30 proteins called nucleoporins that form a symmetrical, 8-fold assembly of spoke-like structures sandwiched between nuclear and cytoplasmic rings, named the NPC core, and filamentous structures that extend into both the nuclear and the cytoplasmic compartments (1,2).
In Saccharomyces cerevisiae, nucleoporins required for mRNA export are found in two subcomplexes of the NPC (3)(4)(5). mRNA export is either blocked or dramatically reduced by mutations affecting most of the nucleoporins in these complexes (5)(6)(7)(8). The strongest mRNA export defects are found in mutants affecting the nucleoporins Nup159p and Nup82p, which are components of the cytoplasmic filaments (5,9). Interactions occur between the C-terminal coiled-coil regions of Nup82p and Nup159p, and Nup159p is lost from NPCs at 37°C in strains where either Nup159p or Nup82p have been truncated to remove part of the coiled-coil region (5,9). A domain at the N terminus of Nup159p provides an important docking site for the essential mRNA export factor Dbp5p/Rat8p, a member of the DEAD box family of RNA helicases (10,11).
The yeast genome encodes more than 30 DEAD box proteins (DBPs), and one or more participate in every aspect of RNA metabolism from synthesis to turnover (reviewed in Refs. [12][13][14]. It is generally believed that DEAD box proteins are RNA helicases, but only a limited number of DBPs have been studied biochemically. Several DBPs have been shown to unwind short double-stranded RNA substrates, and this sometimes requires participation of additional proteins that interact with the DBP (15)(16)(17)(18). Three DEAD box proteins have been shown to mediate dissociation of stably bound proteins from RNA, but the enzymatic properties of most DEAD box proteins have not been determined (19,20).
The precise function performed by the DEAD box protein Dbp5p is not known. Dbp5p interacts directly with the cytoplasmic filaments of the NPC and binds directly not only to Nup159p but also to Gle1p. The filaments also contain another nucleoporin, Nup42/Rip1p (21). Two other proteins that interact with the filaments are Sac3p and Gfd1p (11,(21)(22)(23). The GFD1 gene was identified as high copy suppressor of rat8-2 and gle1-8 mutations and encodes a nonessential coiled-coil protein that interacts with NPCs. Two-hybrid analyses showed that Gfd1p interacts with Gle1p, Dbp5p, and Rip1p/Nup42p (11,21). Recently, Gfd1p has been found associated in a complex with Nab2p both in vivo and in vitro. Nab2p is a shuttling mRNP protein that accompanies the mRNA through the NPC (24). Gfd1p was localized to the cytoplasm and to the nuclear rim (11). Together, these data suggest that Nup159p, Nup82p, Nup42p/Rip1p, Gle1p, Sac3p, and Gfd1p are co-localized at the fibrils of the NPC, and all of them may contribute to the Dbp5p-binding site.
A late step in mRNA export is remodeling of the mRNP complex to remove mRNA-binding proteins needed for mRNA export so that they can return to the nucleus for another round of export. Association of Dbp5p with the cytoplasmic filaments positions it to mediate this remodeling (10,11,21,25). However, Dbp5p also shuttles between the nucleus and cytoplasm (11), and in Chironomus tentans it has been shown that Dbp5p binds to pre-mRNP co-transcriptionally and accompanies the mRNP to and through the nuclear pores (26). An early role for Dbp5p during transcription was also suggested by its genetic and physical interactions with RNA polymerase II components and general transcription factors (27). Thus, Dbp5p could be loaded onto the mRNP early during pre-mRNA biogenesis and may have a later role at the NPC in remodeling the mRNP. By acting on mRNP while bound to the cytoplasmic fibrils of the NPC, Dbp5p may also be able to use the energy of ATP hydrolysis to mediate translocation of the mRNP through the NPC.
Mex67p is thought to play the role of export receptor for mRNA because it shuttles between the nucleus and the cytoplasm, can be cross-linked to poly(A) ϩ in vivo, and interacts directly with the FG repeats of several nucleoporins (revised in Ref. 28). The recruitment of Mex67p to cellular mRNA is facilitated by the mRNA export adaptor Yra1p in association with the RNA helicase Sub2p (29,30). Both Yra1p and Sub2p are recruited to genes in a transcription-dependent manner through a complex called TREX (31).
RNA processing factors are recruited to the elongating mRNA through association with the C-terminal domain of the largest subunit of RNA polymerase II. All pre-mRNA processing steps must be completed accurately before the mRNP can be exported (for a recent review see Ref. 32). In yeast, capping of nascent transcripts at their 5Ј ends is performed by three enzymes: the RNA triphosphatase, Cet1p; the RNA guanylyltransferase, Ceg1p; and the 7-methytransferase, Abd1p. Both Ceg1p and Abd1p bind directly to the phosphorylated C-terminal domain (33). The cap structure is recognized by different proteins in the nucleus and in the cytosol (34,35).
To explore further the role of the Dbp5p in mRNA export, we performed two-hybrid screens to identify additional partners of Dbp5p and its associated factor Gfd1p. We identified Zds1p as a factor interacting with Dbp5p, Gfd1p, and Rip1p. The S. cerevisiae ZDS1 gene and its paralog ZDS2 (zillions of different screens) have been isolated as multicopy suppressors of a diverse array of mutants, including those affecting Cdc42p, a GTPase involved in cell polarity, Cdc28p, a protein kinase required for cell cycle progression, Ceg1p, the RNA guanylyltranferase that participates in cap formation, and Tif1p (yeast eIF4A) (36). ZDS1 and ZDS2 are highly homologous. The homology at the C terminus is particularly striking, with a 100amino acid stretch showing 82% identity. Unfortunately, amino acid sequences of Zds1p and Zds2p are uninformative with respect to its potential function.
Here we report that Zds1p interacts physically with both Dbp5p and Gfd1p, both in vitro and in vivo. Most interestingly, ZDS1/2 mutants have genetic interactions with DBP5, and a zds1⌬/zds2⌬ double mutant is synthetically lethal at 30°C with mex67-5. We also found that the zds1⌬/zds2⌬ double mutant has a moderate defect in mRNA export and that deletion of zds1 and zds2 from dbp5-2 or mex67-5 cells substantially increases their mRNA export defects at temperatures where these mutants are able to grow, and the mRNA export defect is moderate. Reciprocally, we found genetic interactions between DBP5 and both TIF1 and CEG1, two of the genes whose mutations are suppressed by overexpression of ZDS1. Together, the data presented here suggest that Zds1p associates with the complex formed by Dbp5p, Gfd1p, Rip1p/Nup42p, Nup82p, and Nup159p at the cytosolic fibrils of the NPC and is required for optimal mRNA export.

EXPERIMENTAL PROCEDURES
Yeast Plasmid Constructions-The plasmid to express Dbp5p-Gal4p-BD (pDBP5-BD) was obtained by PCR-mediated generation of EcoRI and BamHI sites immediately upstream and downstream, respectively, of the DBP5 coding sequence, and the resulting fragment was ligated into the EcoRI and BamHI sites of pGBDU-C1. Dbp5p-Gal4p-AD was obtained by cloning the same fragment into pGAD-C1 (37). The plasmid for the production of Gfd1p-Gal4p-BD (pGFD1-BD) was obtained by introducing an EcoRI-XhoI fragment containing the complete coding sequence from the GFD1 gene into the EcoRI and SalI sites of pGBDU-C1 (37). The same fragment was inserted into pGAD-C1 to obtain Gfd1p-Gal4p-AD. The plasmids to express the Gle1p, Rip1p-C66, and Rip1p-FG baits and the GST-Dbp5p, GST-Gfd1p, and GST-Rip1p-C66 fusions were the generous gifts from F. Stutz (University of Geneva) and are described previously (21). Fulllength Zds1p and Zds2p fused to the Gal4p-AD were obtained by cloning a PCR fragment containing the entire open reading frame of ZDS1 or ZDS2 into pGAD-C1. YEpTIF1 was generated by PCR amplification of a fragment containing the TIF1 gene (between positions Ϫ500 from the AUG to ϩ457 beyond the stop codon) and insertion between the SacI and PstI sites of YEplac181 (38). YEpSTM1, containing the STM1 gene in the plasmid YEplac181, was a generous gift of P. Linder (Centre Mèdical Universitaire, Universitè de Gèneve).
Strains were grown using standard methods. For growth assays, yeast cells were diluted to an A 600 of 0.1, and serial dilutions (1:10) were spotted onto YDP or selective plates and incubated at various temperatures. Where indicated, 5ЈFOA was added to synthetic complete media at 1 g/liter.
Yeast Two-hybrid Screen-The two-hybrid reporter strain PJ69-4A (37) containing pDBP5-BD or pGFD1-BD was transformed with a set of S. cerevisiae genomic libraries cloned into the activation domain vectors pGAD-C1, pGAD-C2, and pGAD-C3 (37). About 10 6 transformants were plated on synthetic complete medium plates lacking tryptophan, leucine, and histidine and incubated at 30°C for 5 days. Plasmids conferring the ability to grow on plates lacking histidine were isolated from appropriate yeast strains, amplified in Escherichia coli, and rechecked. The region of the yeast genome carried on plasmid inserts was determined by sequencing.
In Situ Poly(A) ϩ RNA Hybridization-Localization of poly(A) ϩ RNA by in situ hybridization with an oligo(dT) 50 probe coupled to digoxigenin was performed as described previously (43), except in Fig. 5A, where a Cy3-end-labeled oligo(dT) 50 was used (44).
In Vitro Binding Assay-GST fusion proteins were produced in E. coli and bound to glutathione-agarose beads as described previously (21). [ 35 S]Methionine-labeled proteins were obtained by coupled T7 transcription-translation in reticulocyte lysates (TNT kit, Promega) as described previously (21). The DNA template for production of Gfd1p was obtained by amplifying yeast genomic DNA with the primers The in vitro binding assays were performed as described previously (21). Immobilized GST fusion proteins were incubated for 1 h at 4°C with the product derived from in vitro transcription/translation. The beads were extensively washed, resuspended in 2ϫ Laemmli sample buffer, and analyzed in 10% polyacrylamide gels. Gels were stained with Coomassie Blue, dried, and scanned using a Fuji FLA-300 Phos-phorImager and Image Reader 1.0 software.

Gfd1p
Interacts in Vitro Directly with Dbp5p-The two-hybrid interaction between Gfd1p and Dbp5p, as reported previously (11), prompted us to determine whether there is a direct association between the two proteins. We isolated a GST-Dbp5p fusion protein produced in E. coli, and we analyzed its ability to interact with Gfd1p, obtained by in vitro translation in a reticulocyte lysate. Fig. 1 shows that 35 S-labeled Gfd1p can be pulled down by GST-Dbp5p bound to glutathione-agarose beads but not by GST alone. This demonstrates that Gfd1p and Dbp5p interact directly.
Identification of Zds1p as a Component of the Dbp5p-Gfd1p Complex-To follow up on our identification of GFD1 as a high copy suppressor of dbp5-2, we performed yeast two-hybrid screens, using Dbp5p-Gal4-BD and Gfd1p-Gal4-BD as baits and an S. cerevisiae genomic library fused to the GAL4 AD. We used a strain where interaction of the two-hybrid proteins is monitored through activation of a HIS3 reporter gene (37).
Among the proteins identified, we recovered Zds1p in both screens. Co-expression of Dbp5-Gal4-BD or Gfd1-Gal4-BD with Zds1-Gal4-AD allowed growth on media lacking histidine, but neither Dbp5-Gal4-BD, Gfd1-Gal4-BD, nor Zds1-Gal4-AD alone was able to activate the HIS3 reporter gene ( Fig. 2A and  results not shown). Although ZDS1 and the closely related ZDS2 have been isolated as multicopy suppressors in many genetic screens (see Refs. 36 and 45), we know much less about their physical interactions, and neither was identified in large scale two-hybrid approaches.
All the Zds1-Gal4-AD fusion proteins identified in the twohybrid screen (seven for Dbp5p and two for Gfd1p) include the C-terminal part of Zds1p (with fusions starting at residues of Zds1p ranging from 530 to 682 (data not shown)), indicating that this is the domain that interacts with Dbp5p and Gfd1p. Most interestingly, this region is highly homologous in ZDS1 and ZDS2 genes, and several observations suggest that the activity of Zds1p may be carried by its C-terminal portion. Thus, truncated alleles of ZDS1, which encodes only amino acids 409 -915 or 796 -915, partially rescue the phenotypes of the zds1 zds2 double mutant strain (45,46). Moreover, these truncated proteins are active as negative regulators of Cdc42p (45) or for dosage suppression of temperature-sensitive mutations affecting the yeast capping enzyme Ceg1p (46). Consistent with this, the C-terminal part of Zds2p (amino acids 781-942) was able to interact with Bcy1p in a two-hybrid assay (54).
By using the two-hybrid system, we were also able to detect an interaction between Gfd1p and the full-length Zds1p, although this interaction was weaker than with the truncated forms of Zds1p ( Fig. 2B and results not shown). Besides the known interaction with Dbp5p, Fig. 2B also shows that Gfd1p interacts with itself and to a lesser extent with Zds2p. Because of the known physical interactions between Dbp5p, Gfd1p, and the fibrils of the NPC (11,21), we examined the interaction of Zds1p with Rip1p and Gle1p. These two proteins are components of the Nup82p subcomplex, and interactions between Gle1p and Dbp5p and between Rip1p and Gfd1p have been reported (11,21). Performing a two-hybrid assay in which the interaction is shown by the appearance of ␤-galactosidase activity, we found that Rip1p but not Gle1p interacts with Zds1p, supporting the idea that Zds1p also interacts with the fibrils of the nuclear pore (Fig. 2C).
Zds1p Interacts with Dbp5p and Gfd1p in Vitro-To determine whether the two-hybrid interactions between Zds1p and both Dbp5p and Gfd1p are direct, we tested their binding in vitro. The C-terminal part of Zds1p, labeled with [ 35 S]methionine, was synthesized in an in vitro transcription-translation system. The in vitro translated protein was incubated with GST fusions to Gfd1p and Dbp5p produced in E. coli and purified on glutathione beads. The C-terminal domain of Zds1p specifically interacted with Gfd1p-GST and Dbp5p-GST but not with Rip1p-GST or GST alone (Fig. 3). None of the GST fusions interacted with in vitro translated luciferase (results not shown; see Ref. 21). These results indicate that Zds1p interacts through its C-terminal domain with both Gfd1p and Dbp5p.
Genetic Interactions between zds1⌬/zds2⌬ and Mutants Affecting mRNA Export Factors-Our results demonstrate a direct physical interaction between Dbp5p and Zds1p. Dbp5p plays a central role in mRNA export, an essential cellular process. Disruption of either ZDS1 or ZDS2 causes only modest phenotypes, and the double zds1⌬//zds2⌬ mutant is viable, although cells have an altered morphology.
To determine whether the function of Dbp5p is affected by the absence of Zds1p, we analyzed the genetic interaction between these genes by constructing a strain lacking ZDS1 (zds1⌬) and containing the temperature-sensitive rat8-2 allele of DBP5. The zds1⌬ rat8-2 double mutant showed impaired growth at semi-restrictive temperatures of 32 and 34°C (data not shown). The impairment was stronger when we also deleted the related ZDS2 gene, suggesting that Zds1p and Zds2p have partially redundant roles with respect to Dbp5p (Fig. 4A). Our data suggest that Zds1p has a positive impact on Dbp5p function.
These findings suggested the possibility that Zds1p and Zds2p might be involved in some aspect of mRNA export. We therefore examined the effect of the absence of ZDS1 and ZDS2 on the growth of a strain carrying a mutant allele of the MEX67 gene. First, we constructed a zds1⌬ mex67-5 double mutant, and we analyzed the growth at different temperatures in comparison with growth of the mex67-5 mutant strain. Fig. 4B shows that deletion of zds1 abolished the ability of the mex67-5 mutant to grow at the semi-restrictive temperature of 34°C. Second, we constructed a strain carrying the zds1⌬ zds2⌬ mutations, a disruption of MEX67, and carrying a wild type MEX67 gene on a URA3/CEN plasmid. This strain was transformed with a LEU2/CEN plasmid containing either wild type MEX67 or the temperature-sensitive mex67-5 allele. Fig. 4C shows that combining zds1⌬ zds2⌬ with mex67-5 resulted in synthetic lethality at 30°C. Therefore, in the absence of both Zds1p and Zds2p, cells producing Mex67-5p as the only form of Mex67p were unable to grow.
Defective Nuclear Export in zds1⌬/zds2⌬ Double Mutants-The genetic interactions between ZDS1 and both DBP5 and MEX67 could reflect an involvement of Zds1p and Zds2p in mRNA export. To analyze mRNA distribution in zds1⌬ zds2⌬ double mutants, we conducted in situ hybridization to localize polyadenylated mRNA. We were able to detect evident accumulation of poly(A) ϩ RNA in nuclei in this strain at low temperature in some cells (Fig. 5A). Although the frequency of nuclear accumulation in the absence of Zds1p and Zds2p is low, we consider it significant, as accumulating cells that showed nuclear accumulation of polyadenylated mRNA were never seen in the isogenic wild type strain.
The requirement of Zds1p and Zds2p for optimal mRNA export was also suggested by exacerbation of the poly(A) ϩ RNA export defects shown by rat8-2 and mex67-5 mutants carrying deletions of ZDS1 or both ZDS1 and ZDS2. Fig. 5B shows that rat8-2 zds1⌬ cells and, to a greater extent, rat8-2 zds1⌬ zds2⌬ cells show substantial nuclear accumulation of poly(A) ϩ RNA, even at room temperature. Moreover, the accumulation of polyadenylated RNA in nuclei in the rat8-2 single mutant at 30 and 34°C was substantially increased in the rat8-2 zds1⌬ and rat8-2 zds1⌬ zds2⌬ strains. Similarly, the poly(A) ϩ accumulation shown by the mex67-5 zds1⌬ double mutant at 34 and 37°C is stronger than in the mex67-5 single mutant (Fig. 5C). Most interestingly, some cells of the mex67-5 zds1⌬ double Further Genetic Links between ZDS1 and DBP5-In previous studies, a diverse set of genes was identified as suppressible by multicopy ZDS1, including the two related to mRNA metabolism and gene expression, CEG1 and TIF1 (46). CEG1 encodes the guanylyltransferase mRNA capping enzyme, and TIF1 encodes the translation initiation factor eIF4A. To determine whether CEG1 and TIF1 could be linked to DBP5 function, we constructed strains carrying mutant alleles of both CEG1 and DBP5, and we found that several ceg1 alleles partially suppressed the growth defect of rat8-2 cells at 34°C (Fig. 6A).
Most interestingly, a genetic connection between DBP5 and TIF1 was found in a genetic screen for multicopy suppressors of the synthetic lethality of the double mutant bur6-1 rat8-2. 2 Fig.  6B shows that the double mutant is able to grow at room temperature when TIF1 is overexpressed, but not in a strain transformed with an empty vector. In a similar way, overexpression of STM1, encoding the yeast translation initiation factor eIF4B (47), also suppresses the synthetic lethality of the bur6-1 rat8-2 double mutant (Fig. 6C).
These findings reveal a web of genetic and physical interactions connecting mRNA export factors (Dbp5p and Mex67p), nucleoporins (Nup159p, Nup82p, Nup42p/Rip1p, and Gle1p), proteins that associate with NPCs (Sac3p and Gfd1p), and translation initiation factors (eIF4A and eIF4B), suggesting that the fibrils of the NPC where Dbp5p binds might have a role in coupling of mRNA export with translation initiation and that Zds1p/Zds2p might facilitate this. In support of this idea, we found that the zds1⌬/zds2⌬ mutant is more sensitive than the isogenic wild type strain to low levels of the translational inhibitor cycloheximide (Fig. 7). DISCUSSION The DEAD box protein Dbp5p is essential for mRNA export, both in yeast and humans (10,25). A fraction of Dbp5p is localized to the cytoplasmic fibrils of the nuclear pore complex, and it has been suggested that this localization would allow Dbp5p to participate in the remodeling of the mRNP complex as the mRNP emerges from the channel of the NPC. However, Dbp5p also shuttles between the nucleus and cytoplasm (11). To avoid futile disassembly of mRNP complexes in the nucleus, it is likely that a mechanism exists to prevent Dbp5p from removing mRNP proteins from the mRNPs when in the nucleus. Because small DBPs like Dbp5p often require co-factors, one way to regulate its activities spatially would be to permit it to associate with co-factors required for this activity only at specific locations such as the fibrils of the NPC.
To better understand the role of Dbp5p in mRNA export, we conducted two-hybrid screens using as baits Dbp5p and a protein identified originally in a search for high copy suppressors of rat8-2, Gfd1p (11). Clones encoding Zds1-GAD fusions were identified multiple times in both two-hybrid screens.
ZDS1 and its paralogue ZDS2 have been identified in a wide variety of genetics screens as multicopy suppressors. This implies that they are involved directly or indirectly in a large number of seemingly unrelated intracellular processes (36). The reason for the broad spectrum of suppression when ZDS1 (and ZDS2) is overexpressed is unknown, but it is not likely that this reflects direct participation of Zds1p in a large number of protein complexes, because only an interaction with Zds2p but not Zds1p was found in a large scale two-hybrid screen (48). The large scale analysis of multiprotein complexes by tandem affinity purification and mass spectrometry only reveals a very small number of partners for Zds1p (49). Specific studies have shown two-hybrid interactions of Zds1p and Zds2p with Sir2p, Sir3p, Sir4p and the yeast telomere-binding protein Rap1p (50) with Bcy1p (54) and the proteins required for cell polarity (51).
Both Dbp5p and Gfd1p interact with the C-terminal portion of Zds1p because this is the only region of the protein present in all of the two-hybrid clones isolated. Furthermore, we showed that the C-terminal portion of Zds1p interacts with Dbp5p and Gfd1p in vitro. However, the interaction can also be observed with the full-length Zds1p. The fact that the interaction with the full-length protein is weaker than with the Cterminal half of Zds1p could reflect Zds1p interacting with multiple partners, some with the N-terminal half and others with the C-terminal half. If a partner that normally bound the N-terminal half were absent (e.g. excluded from the nucleus where two-hybrid interactions take place), the N-terminal part of Zds1p might fold so as to interfere with the ability of the C-terminal part of Zds1p to interact with its binding partners. On the other hand, it is also possible that the binding of partners to the N-terminal part influences (and in this case reduces) the interactions of Zds1p with Gfd1p and Dbp5p.
Although we were not able to localize Zds1p at NPCs, this is the most likely place for interactions of Dbp5p, Gfd1p, and Rip1p with Zds1p to occur because Dbp5p, Gfd1p, and Rip1p are known to interact and to be located at the cytoplasmic fibrils of NPCs. Zds1p might associate only transiently with NPCs and could contribute to creating an optimal binding site for Dbp5p.
In situ poly(A) ϩ RNA hybridization shows that Zds1p and 2 F. Estruch and C. N. Cole, manuscript in preparation. zds1⌬ or zds1⌬ zds2⌬. A and B, the indicated strains (see "Experimental Procedures") were spotted on YPD plates and incubated at different temperatures for 4 days. C, triple mutant mex67-5 zds1⌬ zds2⌬ containing the wild type MEX67 gene in a CEN/URA3 plasmid was transformed with pMEX67/CEN/LEU2 (right) or pmex67-5/CEN/ LEU2 (left) and streaked on 5ЈFOA (upper) and YPD plates (lower). Synthetic lethality was analyzed by incubation at 30°C for 4 days.

FIG. 4. Genetic interactions between rat8-2 and mex67-5 with
Zds2p are not absolutely required for mRNA export. However, at low temperature, when growth of the zds1 zds2 double mutant is compromised (46), a fraction of the cells shows a strong poly(A) ϩ RNA nuclear accumulation that never occurs in the wild type strain. This result could be indicative of a requirement for Zds1/2p in mRNA export only under specific circumstances, such a particular cell cycle stage or in cell aging. However, the results presented in this work suggest a general functional role for Zds1p in mRNA export. We have found that the mRNA export defects of dbp5 and mex67 mutant cells were exacerbated when ZDS1 or both ZDS1 and ZDS2 were disrupted. Moreover, the Hurt laboratory has evidence for a genetic connection between SUS1 and ZDS1, which links Zds1p to another conserved factor involved in mRNA export. 3 Most interestingly, deletion of SUS1 has been found to be synthetically lethal with mutations in DBP5 (52). The requirement of Zds1/2p for mRNA export seems to be absolute when the activity of the export receptor Mex67p is compromised, as shown by the lethality of the triple zds1 zds2 mex67-5 mutant. Because physical interactions suggest that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the NPC, it is tempting to speculate that Zds1p could work by facilitating the interaction of this complex with the proteins bound to the mRNA during its translocation through the NPC. This role could be functionally redundant with other proteins. In this way, it has been suggested recently that Gfd1p, like Zds1p nonessential for export, would act by bridging mRNA export factors and nucleoporins (24).
The proposed role for Dbp5p at the pore in remodeling the protein interaction at the mRNP, together with the overlapping genetic interactions of ZDS1 and DBP5 with both CEG1 and TIF1, suggests a function for the complex in coupling the last steps of mRNA export with translation initiation. To initiate 3 S. Rodriguez-Navarro and E. Hurt, unpublished results.
FIG. 5. Exacerbation of the dbp5 and mex67 poly(A) ؉ export defect by deletion of ZDS1. A, double mutant zds1⌬ zds2⌬ cells were incubated for 4 h at 16°C in YPD, and poly(A) ϩ RNA was visualized by fluorescence in situ hybridization analysis using a Cy3-end-labeled oligo(dT) 50 probe. B and C, the indicated strains were incubated for 1 h at the indicated temperatures. In all cases, cells were fixed, and in situ hybridization was performed using a digoxigenin-conjugated oligo(dT) 50 probe, followed by incubation with a fluorescein isothiocyanateconjugated anti-digoxigenin antibody. RT, room temperature; WT, wild type; DAPI, 4,6-diamidino-2-phenylindole.
FIG. 6. Genetic interactions between dbp5 and genes suppressed by overexpression of ZDS1. A, suppression of rat8-2 growth defect by ceg1 mutations. Double mutant rat8-2 ceg1⌬ cells containing the wild type CEG1 gene or the indicated ceg1 mutant alleles were spotted on YPD plates and incubated at different temperatures for 4 days. B, suppression of the bur6-1 rat8-2 synthetic lethality by overexpression of TIF1. Double mutant bur6-1 rat8-2 containing the wild type DBP5 gene in a CEN/URA3 plasmid was transformed with the multicopy vector YEplac181 empty (YEplac181) or this vector containing the TIF1 gene (YEpTIF1). Transformants were streaked in 5ЈFOA plates and incubated for 5 days at room temperature. C, suppression of the bur6-1 rat8-2 synthetic lethality by overexpression of STM1. Suppression was analyzed as in B but using a YEplac181-derived plasmid containing the STM1 gene (YEpSTM1). translation, the cap-binding protein Cbc20p/Cbc80p is thought to be replaced by eIF4E. Dbp5p might participate in removal of the cap-binding complex, thereby facilitating the transition of the mRNP from export cargo to template for translation. A role for Zds1p as an accessory factor for this process is supported by the increased sensitivity to cycloheximide shown by the zds1⌬ strain. The broad spectrum of mutations suppressed by ZDS1 overexpression could reflect the ability of elevated levels of Zds1p to enhance transfer of mRNA from NPCs to ribosomes, thereby increasing the amount of a mutated protein produced to a level where the mutant strain could grow. A higher level of mutated proteins in cells overexpressing ZDS1 has been reported in some cases (36). This role would also be in agreement with the genetic interaction between DBP5 and TIF1. FIG. 7. Cycloheximide sensitivity of the zds1⌬ zds2⌬ double mutant. Double mutant zds1⌬ zds2⌬ cells and isogenic wild type cells were spotted on YPD plates containing the indicated amounts of the translation inhibitor cycloheximide and incubated for 3 days at 30°C.