The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export.

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rss1p encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rss1p partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37 degrees C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37 degrees C, the mutant Rat7-1p/Nup159-1p is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rss1p did not result in retention of the mutant Rat7-1p/Nup159-1p in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rss1p by placing the RSS1 open reading frame (ORF) under control of the GAL1 promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rss1p are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rss1p overexpression suppresses the growth defect of rat7-1 cells at 37 degrees C by acting to maintain mRNA export.     

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.     

Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes.

To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)50 probe, we identified those that showed nuclear accumulation of poly(A)+ RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT-ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37 degrees C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)+ RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.     

C-terminal truncations of the yeast nucleoporin Nup145p produce a rapid temperature-conditional mRNA export defect and alterations to nuclear structure.

A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)+ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin. NUP145 was previously identified by using a genetic synthetic lethal screen (E. Fabre, W. C. Boelens, C. Wimmer, I. W. Mattaj, and E. C. Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody which recognizes the GLFG family of vertebrate and yeast nucleoporins (S. R. Wente and G. Blobel, J. Cell Biol. 125:955-969, 1994). Cells carrying the new allele, nup145-10, grew at 23 and 30 degrees C but were unable to grow at 37 degrees C. Many cells displayed a modest accumulation of poly(A)+ RNA under permissive growth conditions, and all cells showed dramatic and rapid nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids. This prompted us to examine the role of the carboxyl half of Nup145p. Several additional alleles that encode C-terminally truncated proteins or proteins containing internal deletions of portions of the carboxyl half of Nup145p were constructed. Analysis of these mutants indicates that some sequences between amino acids 698 and 1095 are essential for RNA export and for growth at 37 degrees C. In these strains, nuclear accumulation of poly(A)+ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37 degrees C. Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains. Although cells lacking Nup145p grew extremely slowly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37 degrees C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.     

Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.     

Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene

To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7- 1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p.     

A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.     

A mutation in the gene involved in sister chromatid separation causes a defect in nuclear mRNA export in fission yeast

Fission yeast ptr4-1 is one of the mRNA transport mutants that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature. We cloned the ptr4(+) gene and found that it is identical with the cut1(+) gene essential for chromosome segregation during mitosis. ptr4/cut1 has no defects in nucleocytoplasmic transport of a protein, indicative of a specific blockage of mRNA export by this mutation. A mutant of Cut2p cooperating with Cut1p in sister chromatid separation also showed defective mRNA export at the nonpermissive temperature. Our results suggest a novel linkage between the cell division cycle and nuclear mRNA export in eukaryotic cells.     

The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore

Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC). The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through its conserved N-terminal domain (NTD). Here, we report the crystal structure of the Nup159 NTD, refined to 2.5 A. The structure reveals an unusually asymmetric seven-bladed beta-propeller that is structurally conserved throughout eukarya. Using structure-based conservation analysis, we have targeted specific surface residues for mutagenesis. Residue substitutions in a conserved loop of the NTD abolish in vitro binding to Dbp5, a DEAD box helicase required for mRNA export. In vivo, these mutations cause Dbp5 mislocalization and block mRNA export. These findings suggest that the Nup159 NTD functions in mRNA export as a binding platform, tethering shuttling Dbp5 molecules at the nuclear periphery and locally concentrating this mRNA remodeling factor at the cytoplasmic face of the NPC.     

A hypomorphic allele of the first N-glycosylation gene, ALG7, causes mitochondrial defects in yeast

The modification of proteins at asparagine residues with oligosaccharides (N-glycans) plays critical roles in diverse cell functions. N-glycans originate from a common lipid-linked oligosaccharide (LLO) precursor whose synthesis is initiated by the Dol-P-dependent GlcNAc-1-P transferase (GPT) encoded by an essential ALG7 gene. To identify cellular processes affected by ALG7 and N-glycosylation, we replaced the genomic copy of ALG7 with its hypomorphic allele in two genetically distinct haploid yeast cells. We show that ALG7 knockdown gave rise to an unexpected phenotype of mitochondrial dysfunction. The alg7 mutants did not grow on glycerol and DNA arrays revealed the absence of mitochondrial genes' expression. Accordingly, the alg7 mutants displayed no detectable mtDNA and respiratory activity. Both mutants exhibited diminished abundance of LLO and under-glycosylation of carboxypeptidase Y (CPY). Moreover, another N-glycosylation mutant with a LLO defect, alg6, was respiratory deficient. Collectively, our studies provide evidence that the dysregulation of N-glycosylation in haploid yeast cells leads to mitochondrial dysfunction.     

Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor

TAP-p15 heterodimers have been implicated in the export of mRNAs through nuclear pore complexes (NPCs). We report a structural analysis of the interaction domains of TAP and p15 in a ternary complex with a Phe-Gly (FG) repeat of an NPC component. The TAP-p15 heterodimer is structurally similar to the homodimeric transport factor NTF2, but unlike NTF2, it is incompatible with either homodimerization or Ran binding. The NTF2-like heterodimer functions as a single structural unit in recognizing an FG repeat at a hydrophobic pocket present only on TAP and not on p15. This FG binding site interacts synergistically with a second site at the C terminus of TAP to mediate mRNA transport through the pore. In general, our findings suggest that FG repeats bind with a similar conformation to different classes of transport factors.     

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.     

The fission yeast Nup107-120 complex functionally interacts with the small GTPase Ran/Spi1 and is required for mRNA export, nuclear pore distribution, and proper cell division

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.     

A novel allele of Saccharomyces cerevisiae NDC1 reveals a potential role for the spindle pole body component Ndc1p in nuclear pore assembly

Both the spindle pole body (SPB) and the nuclear pore complex (NPC) are essential organelles embedded in the nuclear envelope throughout the life cycle of the budding yeast Saccharomyces cerevisiae. However, the mechanism by which these two multisubunit structures are inserted into the nuclear envelope during their biogenesis is not well understood. We have previously shown that Ndc1p is the only known integral membrane protein that localizes to both the SPBs and the NPCs and is required for SPB duplication. For this study, we generated a novel temperature-sensitive (ts) allele of NDC1 to investigate the role of Ndc1p at the NPCs. Yeast cells carrying this allele (ndc1-39) failed to insert the SPB into the nuclear envelope at the restrictive temperature. Importantly, the double mutation of ndc1-39 and NPC assembly mutant nic96-1 resulted in cells with enhanced growth defects. While nuclear protein import and NPC distribution in the nuclear envelope were unaffected, ndc1-39 mutants failed to properly incorporate the nucleoporin Nup49p into NPCs. These results provide evidence that Ndc1p is required for NPC assembly in addition to its role in SPB duplication. We postulate that Ndc1p is crucial for the biogenesis of both the SPBs and the NPCs at the step of insertion into the nuclear envelope.     

Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.

In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim.     

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins. Received: 5 September 1996 / Accepted: 21 November 1996