RESULTS

Isolation of synthetic lethal: To identify genes involved in mRNA export, we have isolated mutations that are synthetically lethal when combined with the rae1-167 mutation (Yoonet al. 1997). Four different mutants were isolated that define one complementation group: SL8, SL22, SL27, and SL40. All of these mutants have the same growth and mRNA export phenotypes, and we have used SL27 for this study. The rae1-167 mutant grows normally at 28° with normal poly(A)⁺ distribution (Yoonet al. 1997), but it rapidly accumulates poly(A)⁺ RNA in the nucleus upon a shift to restrictive temperature (>30°). SL27 carries the rae1-167 and the synthetic lethal mutation, sl27, as well as a plasmid (pREP81X-rae1⁺) that expresses the rae1⁺ cDNA from the thiamine repressible nmt81 promoter. The sl27 mutation was separated and backcrossed to rae1-167 to confirm that the synthetic lethality was due to mutation of a single locus. Under permissive conditions when rae1⁺ is expressed in the absence of thiamine, SL27 grows normally at 28°. However, under synthetic lethal conditions when rae1⁺ expression is repressed by the addition of thiamine, SL27 was unable to form colonies at 28° (Figure 1A).

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Figure 1.

—(A) Growth and complementation of the SL27 mutant. SL27 carrying either the pDW232 vector, the full-length nup184 (pUZ184), or the nup184-GFP fusion (pUZ184GFP) plasmids were streaked onto EMM medium in the absence and presence of thiamine (0.5 μm). Plates were incubated for 4 days at 28°. (B) Restriction enzyme map of the nup184 region. The direction of translation, the position of the intron (IVS), and the position of the nonsense nup184-1 mutation (Q960*) are indicated. Below, the region of nup184 that was deleted and replaced by ura4 in the Δnup184 null mutant is indicated. C, ClaI; H, HindIII; K, KpnI; S, SalI; Sa, SacI; Sc, ScaI. (C) Growth and complementation of nup184-1(sl27) on complex medium. The nup184-1 mutants carrying either the pDW232 vector or the full-length nup184 (pUZ184) plasmids were streaked onto nutrient-rich medium containing yeast extract (YES) and grown for 3 days at 28°.

Identification of nup184 as the cognate synthetic lethal gene: To identify the cognate sl27⁺ gene, SL27U (same as SL27 except that rae1⁺ cDNA was expressed from the ura4-based vector pREP82X) cells were transformed with a genomic library with an average insert size of 10 kb in the LEU2-based vector, pWH5 (materials and methods). An 11.5-kb fragment was isolated and subcloned into the ura4⁺ vector, pDW232, from which a 5.9-kb ScaI fragment (pUZ184) was identified that could complement the synthetic lethality of SL27 (Figure 1, A and B). Sequence analysis of this ScaI fragment revealed a large open reading frame that showed a significant similarity to the S. cerevisiae nucleoporin, Nup188p, and analysis of cDNA clones revealed the presence of a single intron. The predicted molecular weight of this protein is 184 kD and showed an overall 23% identity and 48% similarity when compared to Nup188p (Nehrbasset al. 1996; Zabelet al. 1996) of S. cerevisiae (Gen-Bank accession no. AF055035). We named the gene nup184. The nup184 gene contained the sl27 mutation because a SacI-KpnI region was able to marker rescue the synthetic lethality of SL27. Sequence analysis of two independent PCR products using sl27 genomic DNA as template revealed a single mutation that introduces a termination codon at position 960 (Figure 1B). We named the sl27 mutation nup184-1.

nup184 is not essential but is required for growth on nutrient-rich media containing yeast extract: A null allele of nup184 was constructed in a h⁺/h⁺ diploid, SP286, by deletion from amino acid 143 to the end of the coding sequence and insertion of the ura4 gene (Figure 1B). From this diploid, we were able to isolate a haploid Δnup184::ura4 null mutant, indicating that it is not essential for growth (materials and methods). Like nup184-1, the Δnup184 null mutation is synthetically lethal when combined with the rae1-167 mutation, suggesting that nup184-1 is a loss-of-function allele. However, while both nup184-1 and Δnup184 mutants grew well on minimal EMM medium at all temperatures tested (18° to 36°), both grew extremely slowly on nutrient-rich medium containing yeast extract (YES; Figure 1C). This YES sensitivity (YES^S) cosegregates with both the nup184-1 and Δnup184 alleles.

Localization of Nup184p: To determine its localization, Nup184p was tagged at the C terminus with GFP. Expression of this fusion protein from pUZ184GFP was able to complement the synthetic lethality of SL27 (Figure 1A). The Nup184p-GFP fusion was then integrated at the nup184 locus (materials and methods). Fluorescent microscopic examination of the Nup184p-GFP fusion protein revealed its localization to the nuclear periphery in a punctate pattern that is similar to the staining for nuclear pores (Figure 2A). A similar construct fused to full-length Nup184p at the N terminus showed the same localization as the C-terminal fusion. However, the GFP-Nup184-1p fusion, which has N-terminal 960 amino acids, no longer localized to the nuclear periphery (Figure 2A). Also, as with Nup188p (Nehrbasset al. 1996), deletion of the C-terminal 159 amino acids also prevented localization to the nuclear periphery (Figure 2A), and this truncation was unable to complement the synthetic lethality of SL27 (data not shown). On the basis of the sequence similarity to S. cerevisiae Nup188p (Nehrbasset al. 1996; Zabelet al. 1996) and its localization to the nuclear periphery, Nup184p is likely a nucleoporin.

Because the C-terminal truncations of Nup184p did not localize to the nuclear periphery, we tested the nuclear periphery localizations of Rae1p in nup184-1 and Nup184p in rae1-167 mutants under permissive and nonpermissive conditions. Our results show no detectable differences in the nuclear periphery localizations of Nup184p-GFP fusion in rae1-167 at 28° or 36° and the Rae1p-GFP fusion in nup184-1 mutant cells grown in EMM or in YES medium for 12 hr (Figure 2C). These results therefore suggest that the synthetic lethality of the rae1-167 nup184-1 double mutant and the YES sensitivity of nup184-1 is not due to the inability of Nup184p and Rae1p, respectively, to localize to the nuclear periphery.

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Figure 2.

—(A) Localization of Nup184p-GFP fusions. GFP was fused to full-length Nup184p at the C terminus and the construct was integrated at the nup184 locus. The GFP-Nup184-1p and the GFP-Nup184pΔC fusions are truncations of the C-terminal 670 residues and 159 residues, respectively, and both were expressed from a multicopy plasmid. Bar, 10 μm. (B) Thin-section electron micrographs of Δnup184 cells grown in EMM and transferred to YES medium for 24 hr are shown. Nuclear pores are indicated by arrowheads. Bar, 0.5 μm. (C) The effect of rae1-167 and nup184-1 mutations on the nuclear periphery localization of Nup184p-GFP and Rae1p-GFP fusions, respectively. The Nup184p-GFP was integrated at the nup184 locus in rae1-167 cells. These cells were grown at permissive temperature of 28° and shifted to restrictive temperature of 36° for 2 hr. The localization of Rae1p-GFP fusion expressed from a multicopy plasmid was analyzed in nup184-1 cells grown in EMM and shifted to YES for 12 hr at 28°. Coincident 4′,6-diamidino-2-phenylindole (DAPI) staining is shown in the bottom panels.

Because in S. cerevisiae the combination of nup188 with either nic96 or pom152 mutations results in herniations of the nuclear envelope (Nehrbasset al. 1996; Zabelet al. 1996), the morphology of the nuclear envelope of SL27, Δnup184::ura4, and nup184-1 cells was examined by transmission electron microscopy. When the Δnup184 mutants were grown in YES medium, ∼20% of the cells had a dilation of the nuclear envelope when compared to growth in EMM medium (Figure 2B). The distribution and number of pores does not appear to be significantly affected in these mutants. Similar results were seen for SL27 grown in the presence of thiamine and nup184-1 cells grown in YES medium (data not shown). Because the fraction of cells showing gross morphological defects was substantially less than the fraction of cells showing mRNA export defects in these mutants, the gross morphological defects conferred by these mutations cannot account for the mRNA export defects (see below).

mRNA export defects in SL27 and nup184 mutants: To determine whether the growth defects of the nup184 mutants were associated with mRNA export defects, the poly(A)⁺ RNA distribution was examined in SL27, nup184-1, and Δnup184 mutants grown under restrictive conditions. When SL27 (rae1-167 nup184-1/pREP81X-rae1⁺) was grown in the absence of thiamine, distribution of the poly(A)⁺ RNA in the cells was normal (Figure 3A), but within 12 hr after the addition of thiamine and repression of rae1⁺ expression, >90% of the cells showed strong nuclear accumulation of poly(A)⁺ RNA. Under similar conditions, the poly(A)⁺ distribution of RNA in rae1-167 was normal (Figure 3A; Yoonet al. 1997). Therefore, the synthetic lethality is associated with a defect in the export of mRNA from the nucleus. When the nup184-1 mutant was grown in EMM, <5% of the cells had nuclear accumulation of poly(A)⁺ RNA. Within an hour after a shift from EMM medium to nutrient-rich YES medium, nuclear accumulation of poly(A)⁺ RNA was detected in ∼25% of the cells (data not shown). As incubation continued, both the fraction of cells and the extent of nuclear accumulation increased. By 12 hr, 75% of the nup184-1 cells had strong nuclear accumulation of poly(A)⁺ RNA (Figure 3A). Additionally, the Δnup184 cells showed a stronger mRNA export defect when grown in EMM where 50% of the cells had nuclear poly(A)⁺ RNA accumulation. Incubation of Δnup184 cells in YES for 12 hr increased the strength and extent of nuclear poly(A)⁺ RNA accumulation to a level similar to that seen for nup184-1 cells grown in YES (Figure 3A). Taken together, these results suggest that Nup184p has a role in mRNA export from the nucleus.

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Figure 3.

—(A) Poly(A)⁺ distribution in SL27, nup184-1, and Δnup184::ura4 mutants. Cells were grown to mid-log phase in appropriately supplemented EMM medium in the absence of thiamine (–B1) at 28°. SL27 cells were then shifted to EMM medium containing thiamine (+B1) and grown for 24 hr. The nup184-1 and Δnup184::ura4 mutants were shifted to YES medium and grown for 12 hr at 28°. Cells were fixed and the poly(A)⁺ RNA was visualized by fluorescent in situ hybridization. Coincident DAPI staining is shown in the bottom panels. Bar, 10 μm. (B) Nuclear protein import analysis. Plasmids expressing the spMex67-NLS-GFP reporter protein expressed from the mex67 promoter were introduced into wild-type, SL27, Δnup184, and nup184-1 cells. SL27 cells were shifted to EMM medium containing thiamine (+B1) for 12 hr, and Δnup184 and nup184-1 cells were transferred to YES medium for 12 hr before in vivo nuclear import analysis was performed. spMex67-NLS-GFP reporter protein was visualized by fluorescent microscopy. Top panels (–energy) show diffusion of reporter protein in cells treated with 10 mm sodium azide and 10 mm deoxyglucose for 45 min. Bottom panels (+energy) show reimport of the reporter protein into the nucleus after cells were washed and resuspended in glucose-containing medium for 15 min to regenerate energy.

To determine whether another NPC function is affected in nup184 mutants, nuclear protein import of a SV40-NLS-GFP-LacZ reporter was examined (data not shown). In wild-type cells, in SL27 grown in the presence and absence of thiamine, as well as in Δnup184 and nup184-1 cells grown in EMM and YES medium, this reporter was localized exclusively to the nucleus with no reporter detected in the cytoplasm. As a control, the ptr3-1 mutant, which has a defect in protein import (Azadet al. 1997), accumulated a significant amount of the reporter protein in the cytoplasm within 3 hr after a shift to restrictive temperature. Because poly(A)⁺RNA export is inhibited in SL27 and nup184 mutants under restrictive conditions, we cannot exclude the possibility that the nuclear reporter protein is no longer synthesized under restrictive conditions due to cytoplasmic depletion of poly(A)⁺RNA. We also used a recently developed method for monitoring protein import that is not dependent on continued mRNA synthesis and export (Shulgaet al. 1996). A spMex67-NLS-GFP fusion was expressed from a multicopy plasmid in wild type, SL27 grown in absence and presence of thiamine for 12 hr, and nup184-1 cells grown in EMM and in YES medium for 12 hr. This reporter localized predominantly in the nucleus (data not shown). However, when these cells were depleted for energy by treatment in glucose-free medium containing sodium azide and deoxyglucose, the low-molecular-weight reporter protein diffused into the cytoplasm (Figure 3B). After 45 min the cells were washed and returned to glucose-containing medium to restore energy. This allowed rapid reimport of the spMex67-NLS-GFP reporter in all cells within 15 min (Figure 3B). These results suggest that at least some of the transport functions of the NPC remain intact, and the mRNA export defect is not the result of the loss of pore function in general.

Sensitivity to growth on YES is responsive to a cAMPdependent pathway: To understand the basis for the YES^S of the nup184 mutants, we isolated a multicopy suppressor of the YES^S of nup184-1. Sequence analysis of a clone, pKYR, revealed that it carried a gene, cgs2, encoding a cAMP-phosphodiesterase (Devotiet al. 1991). This raised the possibility that mRNA export is regulated by a cAMP-dependent pathway, and this regulation is revealed by the nup184 mutants. If activation of the cAMP-dependent pathway is necessary for the mRNA export defect, then inactivation of the pathway should allow growth of nup184-1 and suppress the mRNA export defect on YES. Therefore, nup184-1 was crossed with Δpka1, defective for the cAMP-dependent protein kinase in S. pombe, Pka1p (Yuet al. 1994). Indeed, removal of Pka1p relieved the growth defect and poly(A)⁺ RNA export defect of the nup184-1 mutant when grown in YES (Figure 4, A and B). Moreover, inactivation of the cAMP-dependent pathway in SL27 suppressed the growth defect (Figure 4A) and mRNA defects of the synthetic lethality (Figure 4B). Because

nup184-1 has partial activity (the Δnup184 mutant has a stronger mRNA export defect when grown on EMM;

Figure 3A), it was possible that Δpka1 increased the activity of Nup184-1p, and this was responsible for the suppression. However, Δpka1 was also able to suppress

the growth defect of Δnup184 in YES (Figure 4A) and rescue the poly(A)⁺ RNA export defect of Δnup184 in EMM and in YES medium (compare Figure 4B to Figure 3A), suggesting that the Pka1p kinase pathway modulates a function other than Nup184p. Interestingly, there were some residual poly(A)⁺ RNA export defects when Δpka1 nup184 mutants were grown in YES, leaving open the possibility that nup184 function could be regulated in part by nutritional status.

To test whether activation of the Pka1p kinase was sufficient to block mRNA export in nup184-1 cells, a Δcgs1 null mutation, removing the negative regulatory subunit of Pka1p (Devotiet al. 1991), was introduced into nup184-1 cells. Indeed, the poly(A)⁺ RNA export defects of the Δcgs1 nup184-1 mutant were significantly higher in EMM medium when compared to individual mutants grown under similar conditions (Figure 4B). However, the poly(A)⁺ RNA export defect of the Δcgs1 nup184-1 mutant in EMM is not as severe as in YES (Figure 4B). Interestingly, the Δcgs1 mutation itself conferred a modest nuclear poly(A)⁺ RNA accumulation in nearly every cell (Figure 4B), suggesting that the cAMP pathway can downregulate mRNA export even in a nup184⁺ background. Similar results were obtained when the Pka1 kinase pathway was activated by exogenous addition of 10 mm cAMP (8-bromo-cAMP) to wildtype cells grown in EMM (data not shown). However, addition of cAMP to wild-type cells, similar to Δcgs1 mutant, did not increase the poly(A)⁺ RNA accumulation in the nucleus following shift into YES medium. Taking these results together, we conclude that the cAMP-dependent signal transduction pathway is necessary but not sufficient for the amount of export defect seen in nup184 mutants grown in nutrient-rich medium. Therefore, there is another pathway that senses nutritional status also contributing to the downregulation of mRNA export.

The mRNA export defect of nup184-1 in nutrient-rich medium is a post-translational event: Because cAMP levels and nutritional status regulate gene expression, the possibility exists that the mRNA export defect seen in nup184-1 is a result of lowered expression of a labile mRNA export factor that is required in the absence of nup184 function. However, addition of cycloheximide to nup184-1 cells grown in EMM did not affect poly(A)⁺ RNA distribution (Figure 5). Moreover, addition of cycloheximide did not affect either the rate or extent of nuclear poly(A)⁺RNA accumulation following a shift of nup184-1 cells to YES for 4 hr (Figure 5). These results show that the regulation of mRNA export by nutritional status (YES) is a post-translational event. However, addition of cycloheximide blocked nuclear poly(A)⁺ accumulation following the addition of cAMP to nup184-1 cells grown in EMM (Figure 5). Thus, the regulation by the cAMP-dependent Pka1p kinase is a translation-dependent event. This result and that of the Δpka1 null suppression of poly(A)⁺ RNA export defect in Δnup184 mutant in EMM and in YES medium suggest that Pka1p kinase activity is required for the expression of a factor necessary for the inhibition of mRNA export. While we cannot rule out nutritional regulation of the stability of an mRNA export factor, we favor a hypothesis in which a nutritional signal transduction pathway directly or in-directly regulates nucleocytoplasmic trafficking by modifying the transport machinery. We conclude from our results that activation of the Pka1p pathway is necessary to negatively regulate poly(A)⁺ RNA export defect, but is not sufficient to cause the severe poly(A)⁺ RNA export defects seen in nup184-1 cells grown in nutrient-rich medium.

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Figure 4.

—Growth of nup184, cgs1, and pka1 mutants on minimal and nutrient-rich media. (A) Serial dilutions of cells were spotted onto appropriately supplemented minimal EMM and nutrient-rich YES medium and grown for 5 days and 3 days, respectively. (B) Poly(A)⁺ RNA distribution in nup184, cgs1, and pka1 mutant cells as indicated. Cells were grown to log phase in EMM and shifted to YES medium followed by a 12-hr incubation. Cells were fixed and poly(A)⁺ RNA was localized by fluorescent in situ hybridization.