Title: Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells
Abstract: Article15 October 1999free access Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells Christine A. Hodge Christine A. Hodge Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Hildur V. Colot Hildur V. Colot Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Phillip Stafford Phillip Stafford Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Charles N. Cole Corresponding Author Charles N. Cole Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Christine A. Hodge Christine A. Hodge Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Hildur V. Colot Hildur V. Colot Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Phillip Stafford Phillip Stafford Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Charles N. Cole Corresponding Author Charles N. Cole Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA Search for more papers by this author Author Information Christine A. Hodge1, Hildur V. Colot1, Phillip Stafford1 and Charles N. Cole 1 1Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755 USA ‡C.A.Hodge and H.V.Colot contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:5778-5788https://doi.org/10.1093/emboj/18.20.5778 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 (Rat7pΔN) 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 rat7ΔN 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. Introduction Transport of macromolecules between nucleus and cytoplasm is an essential process in eukaryotic cells (for review see Corbett and Silver, 1997; Ohno et al., 1998) and takes place through nuclear pore complexes (NPCs). NPCs are large proteinaceous complexes embedded in the nuclear envelope and contain (in yeast) ∼30 proteins called nucleoporins. Through a genetic approach, we determined that several nucleoporins function specifically in mRNA export (Gorsch et al., 1995; Heath et al., 1995; Li et al., 1995; Goldstein et al., 1996; Dockendorff et al., 1997). Transport also requires soluble receptors, which recognize signals in molecules to be transported, as well as the activities of Ran (Gsp1p in yeast), a small ras-like GTPase, and its effectors (Rna1p and Prp20p). These components function to ensure that cargoes are transported only in the proper direction (for review see Gürlich, 1998). The known receptors are all related to importin β (Kap95p/Rsl1p in yeast). They share a Ran-binding domain and interact with the FG repeat regions of nucleoporins. Ran regulates the assembly and disassembly of complexes between transport receptors and their cargoes. The export of mRNA to the cytoplasm appears to be considerably more complex than transport of smaller cargoes, including proteins and tRNAs (for review see Stutz and Rosbash, 1998). mRNAs are exported as mRNP particles, which contain mRNA and several RNA-binding proteins. These proteins may function to package the mRNA in an exportable configuration and may also directly mediate interactions with other transport factors (for review see Nakielny and Dreyfuss, 1997). Several additional proteins required for mRNA export have been identified through genetic and biochemical approaches; most are highly conserved between yeast and metazoans. These include Mex67p (TAP in human cells) (Segref et al., 1997), Gle1p/Rss1p/Brr3p (hGLE1 in human cells) (Del Priore et al., 1996; Murphy and Wente, 1996; Noble and Guthrie, 1996a; Watkins et al., 1998), Gle2p (Rae1p in Schizosaccharomyces pombe) (Brown et al., 1995; Murphy et al., 1996) and Mtr2p (Santos-Rosa et al., 1998; Katahira et al., 1999). All are essential, and yeast strains carrying temperature-sensitive (ts) mutations in any of these genes show rapid and strong defects in mRNA export. Interestingly, none of these factors is required for protein transport. Transport receptors and Ran/Gsp1p shuttle continuously between nucleus and cytoplasm. Mex67p and Mtr2p have also been shown to shuttle (Segref et al., 1997; Santos-Rosa et al., 1998), as do several of the RNA-binding proteins that exit the nucleus as part of the mRNP. The specific functions and mechanisms of action of these export factors have not yet been determined. The only export receptor of the importin β family that shows defects in mRNA export when mutated is Crm1p/Xpo1p. Xpo1p mediates the export of proteins containing Leu-rich nuclear export signals (LR-NES) (Stade et al., 1997). Although poly(A)+ RNA accumulates in nuclei of xpo1-1 cells at 37°C, it is not known whether this reflects a direct or indirect role for Xpo1p in mRNA export. The finding that protein synthesis continues at a normal level when cells carrying a leptomycin B-sensitive allele of XPO1 are treated with leptomycin B is consistent with the idea that mRNA export continues even when Xpo1p is not functional (Neville and Rosbash, 1999). The same genetic screen we used to identify RNA export-specific nucleoporins also identified a DEAD-box protein, Dbp5p/Rat8p (Snay-Hodge et al., 1998). DEAD-box proteins are thought to use the energy derived from ATP hydrolysis to unwind RNA secondary structure and to alter RNA-protein interactions. Multiple DEAD-box proteins are found in all organisms from bacteria to mammals. Approximately 28 have been identified in yeast. Biochemical and genetic studies indicate that DEAD-box proteins play an essential role in most steps of RNA metabolism from synthesis to turnover (for review see de la Cruz et al., 1999). Dbp5p is essential for mRNA export. In strains carrying ts alleles of DBP5, poly(A)+ RNA accumulates in nuclei of all cells rapidly (<10 min) and synchronously upon a shift to 37°C (Snay-Hodge et al., 1998; Tseng et al., 1998). Dbp5p is very abundant, has ATP-binding and ATPase activities, and also shows RNA-unwinding activity but only when assayed in the presence of a yeast cell extract (Tseng et al., 1998). Most Dbp5p is cytoplasmic, but a fraction associates with NPCs (Snay-Hodge et al., 1998). The N-terminal region (amino acids 9-79) of Dbp5p is not essential (Snay-Hodge et al., 1998). In this paper, we show that Dbp5p and Rat7p/Nup159p interact and this association requires their N-termini. Rat7p is one of several nucleoporins essential for mRNA export and is not required for transport of proteins or export of tRNA (Sarkar and Hopper, 1998) and small ribosomal subunits (Moy and Silver, 1999). We also show that Dbp5p interacts with the export factor Gle1p, shown previously to be associated with NPCs (Del Priore et al., 1996; Murphy and Wente, 1996). In addition, we report that Dbp5p shuttles between nucleus and cytoplasm. Dbp5p accumulates in nuclei of xpo1-1 cells, and also in cells carrying mutant alleles of MEX67, RNA1, PRP20 or GSP1 (Ran). Overexpression of Dbp5p prevents mRNA accumulation in xpo1-1 cells shifted to 37°C. This supports the idea that Xpo1p may not play an essential role in mRNA export. Finally, we have conducted a screen to identify high-copy suppressors of the rat8-2 allele of DBP5. Through this we identified YMR255w, which encodes a non-essential 21 kDa protein of unknown function, located in the cytoplasm with some accumulation at the nuclear rim, a pattern similar to that of Dbp5p. Results Overexpression of Dbp5p suppresses the growth and export defects of rat7 mutant strains We are interested in defining the specific functions that Dbp5p and Rat7p play in mRNA export. In the experiments presented here, we used the three mutant alleles of RAT7 described in earlier publications. One is the original rat7-1 allele, which encodes a protein truncated within its C-terminal third, removing one of two adjacent coiled-coil regions (Gorsch et al., 1995). rat7ΔR and rat7ΔN encode proteins lacking the central repeat domain or the N-terminal third of Rat7p, respectively (Del Priore et al., 1997). The central repeat domain is not essential whereas growth becomes temperature sensitive when the Nterminus is deleted (Del Priore et al., 1997). There is greater accumulation of poly(A)+ mRNA in nuclei of rat7ΔN cells at 23°C than in rat7-1 cells, and a complete export block at 37°C. Rat7-1p is lost from NPCs at 37°C, but Rat7pΔN is not. We tested whether overexpression of Dbp5p would affect the growth and mRNA export defects of rat7 mutants. As a control, we overexpressed Gle1p, which we identified previously as a high-copy suppressor of rat7-1 (Del Priore et al., 1996). Compared with Gle1p, overexpressed Dbp5p was a better suppressor of the 37°C growth defect of rat7-1 cells (Figure 1). Dbp5p enhanced mRNA export in rat7-1 cells (Figure 2; compare B and H), but nuclear accumulation of poly(A)+ RNA was still readily detected, similar to the effect of overexpression of Gle1p on mRNA export (Del Priore et al., 1996). Overexpressed Dbp5p (Figure 1), but not Gle1p (data not shown), was also able to suppress the growth defect of rat7ΔN cells at 37°C (Figure 1) and completely prevented accumulation of poly(A)+ RNA in rat7ΔN nuclei at both 23 and 37°C (Figure 2I and J). In addition, Dbp5p was able to suppress the growth defect of rss1-37 cells (Figure 1), but Gle1p could not suppress the rat8-2 growth defect (data not shown). Modestly-reduced nuclear and increased cytoplasmic levels of poly(A)+ RNA were observed when Dbp5p was overexpressed in rss1-37 cells (Figure 2, compare F and L). The 2μ plasmids used for these studies result in an ∼8-fold increase in Dbp5p (Snay-Hodge et al., 1998) and an ∼5-fold increase in Gle1p levels (Del Priore et al., 1996). Figure 1.Suppression of ts growth defects by overexpression of Dbp5p. Yeast strains carrying the rat7-1, rat7ΔN or rss1-37 alleles were transformed with high-copy (2μ) plasmids expressing Dbp5p or Gle1p. As a control, the same strains were transformed with the vector. Strains were grown on selective media, diluted to OD600 of 0.05, and 1:5 serial dilutions were spotted onto plates, which were incubated for 4 days at the temperatures indicated. Download figure Download PowerPoint Figure 2.Over-expression of Dbp5p suppresses the mRNA export defect of rat7ΔN cells. rat7-1, rat7ΔN and rss1-37 cells were transformed either with a high-copy (2μ) plasmid expressing Dbp5p or with empty vector. Cells were analyzed by in situ hybridization using an oligo(dT)50 probe. Download figure Download PowerPoint Dbp5p interacts with the N-terminal domain of Rat7p These results suggested that Dbp5p might interact physically with Rat7p. Dbp5p was able to pull down wild-type Rat7p (Figure 3A, lane 1) and Rat7pΔR (Figure 3B, lane 4), but not Rat7pΔN (Figure 3B, lane 2). Rat7p is unstable under the extraction conditions used, explaining the breakdown products seen in this experiment. Dbp5p also showed a strong interaction with the N-terminal third of Rat7p when the latter was expressed as a separate polypeptide (Figure 3C, lane 3). In an attempt to determine which portions of Dbp5p interact with Rat7p, we tested two N-terminally-deleted mutants. A greatly reduced interaction was seen when amino acids 9-79 of Dbp5p were deleted (Figure 3A, lane 2) even though this allele supports growth without detectable mRNA-export defects (Snay-Hodge et al., 1998). No interaction was detected between Rat7p and Dbp5pΔ9-122 (Figure 3A, lane 3); Dbp5pΔ9-122 does not support growth. Western blot analyses demonstrated that the Dbp5pΔ9-79 and Dbp5pΔ9-122 proteins were present at high levels in the extracts used (data not shown). We were unable to co-immunoprecipitate Rat7p with only the N-terminal portion of Dbp5p (aa1-122) (data not shown), indicating that this region is necessary but not sufficient for the interaction between Rat7p and Dbp5p. Figure 3.Western analysis of proteins pulled down with Dbp5p-His. (A) Strains harboring His-tagged wild-type (WT) or N-terminally deleted Dbp5p were subjected to pull-down and the eluted proteins analyzed with an anti-Rat7pR antibody. Rat7p is highly susceptible to degradation under the extraction conditions used (Gorsch et al., 1995). (B) His-tagged wild-type Dbp5p was expressed in strains containing deletion alleles of RAT7. Bound proteins were analyzed as in (A), except that an anti-Rat7pN antibody was used for lanes 3 and 4. (C) His-tagged Dbp5p and Rat7pN were co-expressed in a wild-type strain and subjected to pull-down analysis as in (A), except that the anti-Rat7pN antibody was used. (D) His-tagged Dbp5p, either wild-type or N-terminally deleted, was expressed in strains containing ProtA-Gle1p and subjected to pull-down analysis. An anti-protein A antibody was used to detect ProtA-Gle1p. Download figure Download PowerPoint Additional biochemical analyses also indicated that Dbp5p and Gle1p interact with one another (Figure 3D, lane 2). Dbp5pΔ9-79 and wild-type Dbp5p interacted equally well with Gle1p (Figure 3D, compare lanes 3 and 4). However, little or no interaction was seen between Gle1p and Dbp5pΔ9-122 (data not shown). It is not possible from these studies to determine if the interactions are direct or mediated by other proteins. Two-hybrid analyses were also used to examine these interactions. The data summarized in Table I indicate a strong interaction of the N-terminal third of Rat7p with Dbp5p, a somewhat weaker interaction with Dbp5pΔ9-79, and little or no interaction with Dbp5pΔ9-122. No interactions were seen between Dbp5p and any of the other nucleoporins tested. Dbp5p could not interact with the central repeat region of Rat7p (Table I), but a strong two-hybrid interaction was seen between this region of Rat7p and Xpo1p. Equally strong interactions were seen between Xpo1p and the repeat regions of Nup100p and Rip1p/Nup42p, as well as human CAN/NUP214 and NUP98, consistent with earlier results of others (Neville et al., 1997). It was not possible to isolate cells expressing full-length Gle1p in a two hybrid construct (F.Stutz, personal communication) so we used a construct expressing only the C-terminal half (257-538), missing the non-essential N-terminal region and the essential coiled-coil domain (Del Priore et al., 1996). This portion of Gle1p interacted strongly with Dbp5p. We conclude that Dbp5p can interact with the N-terminal part of Rat7p and with the C-terminal half of Gle1p. Table 1. Yeast two-hybrid interactions between Dbp5p, nucleoporins and transport factors β-galactosidase activitya Prey Bait Dbp5p WT Dbp5p (Δ9-79) Dbp5p (Δ9-122) Rat7p (1-500) Xpo1p WT Gle1p (257-538) Gfd1p WT Dbp5p WT − − − ++ − +++ + Rat7p repeats (497-701) XXFG − +++ + − Rat7p N-terminus (1-500) +++ ++ − − Nup1p N-terminus (1-488) − − CAN/NUP214 repeats (1691-1894) XXFG − − − − +++ +++ − Rip1p repeats (121-230) XXFG − − − − +++ + +++ Nup100p repeats (278-539) GLFG − − − − + − − Human NUP98 repeats (41-515) GLFG − − − − ++ ++ − Nup1p repeats (438-737) XFXFG − − − − − − − Gle1p (257-538) − − − − − − + Gfd1p + − − − − +++ − a β-galactosidase production was measured in diploid strains obtained by mating haploids containing bait and prey constructs. Three distinct types of nucleoporin repeats (XXFG, GLFG, XFXFG) were used along with Xpo1p and the N-terminal non-repeat region of Nup1p as controls to verify the specificity of the interactions detected. The positive control (a LexA DNA-binding domain fused to a GAL4 activation domain) gave 6000-8000 Miller units (Miller, 1972) while the negative controls (empty prey vector pJG4-5 along with various baits) gave 50-180 Miller units. Key: +++, >6000 Miller units; ++, 4000-6000 Miller units; +, 200-4000 Miller units; −, <200 Miller units. Expression of high levels of Rat7p fragments reduces growth and mRNA export Next we investigated the effects on growth and mRNA export of inducing high-level expression of either the N-terminal region or the central repeat domain of Rat7p, using the inducible GAL1 promoter. The Western blot in Figure 4A shows that the levels of these fragments were very low (Rat7pN) or nil (Rat7pR) in the absence of galactose. After galactose induction, both Rat7p fragments accumulated to very high levels compared with the level of endogenous Rat7p. After 36 h in galactose, Rat7pN caused a modest accumulation of poly(A)+ RNA in nuclei (Figure 4B, compare panels A and C), but no defects in growth. This minor defect was suppressed by overexpression of Dbp5p in these cells (Figure 4B, panel E). Figure 4.Expression of high levels of Rat7pN or Rat7pR in wild-type cells. (A) Extracts were prepared from cells expressing either Rat7pN (aa1-456) or Rat7pR (aa457-899) under an inducible GAL promoter. Extracts made either prior to (right lanes) or following induction with 2% galactose for 1 h (left lanes) were analyzed by Western blotting. The position within the gel of the endogenous wild-type Rat7p is indicated. (B) The effect of high-level expression of Rat7pN on mRNA export was determined by in situ hybridization (A, C, E); panels B, D and F show DAPI staining of the same cells as are shown in panels A,C and E, respectively. Panels A and B, cells grown in dextrose; panels C and D, cells grown for 36 h on 2% galactose; panels E and F, cells containing a high-copy DBP5 plasmid, shifted to 2% galactose for 36 h. (C) Inhibition of growth of wild-type cells by Rat7pR and its suppression by high-copy XPO1. Serial dilutions (1:5) of wild-type cells carrying a 2μ plasmid containing either no insert (vector), DBP5 or XPO1 were plated on dextrose (left panel) or galactose (three right panels) and incubated at the temperatures shown for 3 days. Download figure Download PowerPoint In contrast, Rat7pR impaired growth of wild-type cells at 23 and 30°C (Figure 4C), whereas normal growth was seen at 37°C. However, mRNA export was unaffected by high-level expression of Rat7R (data not shown). Overexpression of Dbp5p had no effect on the growth inhibition caused by high-level expression of Rat7pR, whereas expression of Xpo1p from a high-copy plasmid suppressed this growth defect at 30°C, but not at 23°C (Figure 4C). The most likely explanation for this is that the loss of the dominant-negative effect of Rat7pR at 37°C reflects an interaction between Rat7pR and Xpo1p that is weaker than that which occurs during the probable interactions of the full-length proteins at the NPC. Taken together, these data are consistent with overexpressed Rat7pN titrating Dbp5p and overexpressed Rat7pR titrating Xpo1p. The N-terminal domain of Rat7p is essential for association of Dbp5p with NPCs In earlier studies we demonstrated that Dbp5p was located primarily in the cytoplasm with a fraction associated with NPCs (Snay-Hodge et al., 1998). The data described above suggest that the N-terminal portion of Rat7p is important for this interaction. Therefore, we examined the location of a fully functional Dbp5p-GFP (green fluorescent protein) fusion in rat7ΔN cells as well as in nup82Δ108 cells, in which Rat7p dissociates from NPCs when cells are shifted to 37°C (Belgareh et al., 1998). At 23°C Dbp5p-GFP associated with NPCs as well as wild-type Dbp5p in nup82Δ108 cells (Figure 5A). As reported previously, Dbp5p associated with clustered NPCs in the rat2-1 strain (Snay-Hodge et al., 1998). In contrast, in rat7ΔN cells, Dbp5p was not detected at the nuclear rim at either 23 or 37°C. Dbp5p could be detected at the nuclear rim in rat7-1 cells incubated at 23°C, but the intensity of the signal was much lower than for other strains. Upon shifting cells to 37°C, we observed no change in the intensity of nuclear rim-associated Dbp5p-GFP in wild-type or rat2-1 cells. Rat7p dissociated from NPCs in both rat7-1 and nup82Δ108 cells shifted to 37°C. These data indicate that the N-terminal portion of Rat7p is essential for association of Dbp5p with NPCs. Figure 5.Localization of Dbp5p in strains carrying mutations affecting mRNA export. Dbp5p-GFP was observed in living cells by spotting overnight cultures onto coverslips coated with 1% polylysine. Strains were shifted to 37°C for 30-150 min (based on the times previously determined to be necessary for maximal mRNA export defects to occur), then spotted onto prewarmed coverslips and slides and photographed using either a Zeiss Axioplan B (A) or Axiophot (B), both equipped with a cooled CCD camera. In (A) a z-series was taken of each strain with 0.2 μm increments, deconvolved using OpenLab software (Improvision, Conventry, UK) and then 3D rendered. In (B) single slices that included the nucleus were photographed, and no deconvolution was performed. Download figure Download PowerPoint Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner Shuttling across the nuclear envelope is a property of many factors required for mRNA export, including transport receptors and Gsp1p (Ran). To determine whether Dbp5p shuttles, we examined the location of Dbp5p-GFP in xpo1-1 cells. Dbp5p was detected primarily in nuclei at 23°C, but a cytoplasmic signal was clearly evident. Following a shift to 37°C, Dbp5p was detected only in nuclei (Figure 5B), a condition where Xpo1p-dependent protein export is blocked. This indicates that Dbp5p is a shuttling protein. When cells were returned to 23°C, most of Dbp5p-GFP was exported from nuclei within 1 h (data not shown). Dbp5p accumulates in nuclei in several strains with ts defects in nuclear transport The behavior of Dbp5p in xpo1-1 cells led us to analyze the distribution of Dbp5p in cells carrying mutations affecting other soluble mRNA transport factors. At 37°C, strong nuclear accumulation of Dbp5p was seen in cells that harbor mutant alleles of the components of the Ran-GTPase system (rna1-1, prp20-1 or gsp1-1) (Figure 5B). Note that a cytoplasmic signal for Dbp5p can be seen in all of these strains except rna1-1. Dbp5p accumulated in nuclei of yrb2Δ cells shifted to 16°C for 1 h (data not shown). Yrb2p is one of the few transport factors identified that is important for protein export (Noguchi et al., 1997; Taura et al., 1998); cells with a disruption of YRB2 are cold-sensitive for both growth and protein export. Interestingly, mutation of MEX67 (mex67-5) also caused nuclear accumulation of Dbp5p, whereas mutation of GLE1 (the rss1-37 allele) or GLE2 (data not shown) did not. To assess the specificity of these effects, we examined the location of Dbp5p in other mutant strains. Dbp5p was distributed normally in strains carrying mutations affecting several nucleoporins required for mRNA export (RAT7/NUP159, RAT2/NUP120, RAT3/NUP133, RAT9/NUP85 and NUP145/RAT10), in two strains with mutations of protein synthesis initiation factors (tif1-1 and prt1-1) and in strains disrupted for two non-essential importin β family members (msn5Δ or sxm1Δ). Tif1p encodes the DEAD-box protein eIF4A; Prt1p is a component of eIF3. Overexpression of Dbp5p prevents accumulation of poly(A)+ RNA in xpo1-1 cells When xpo1-1 cells are shifted to 37°C, poly(A)+ RNA accumulates in nuclei (Stade et al., 1997; Figure 6A). Although overexpression of Dbp5p could not suppress the growth defect of xpo1-1 cells (data not shown), it completely prevented accumulation of poly(A)+ RNA in these cells (Figure 6A, compare panels C and G). We localized overexpressed Dbp5p in xpo1-1 cells, and observed cytoplasmic Dbp5p both at 23°C and following a shift to 37°C (Figure 6B). At 23°C, all cells showed primarily cytoplasmic Dbp5p, with little or no nuclear signal; in addition, over-expression of Dbp5p enhanced the growth of xpo1-1 cells at 23°C. Following the shift to 37°C, all cells showed both nuclear and cytoplasmic Dbp5p. Cytoplasmic Dbp5p was most abundant in cells expressing the highest levels of Dbp5p. This suggests that Dbp5p acts within the cytoplasm to prevent an mRNA export block in these cells. To make certain that mRNA continued to be produced and exported following the shift to 37°C, we induced expression from a GAL promoter and analyzed RNA accumulation by Northern blot analysis. No RNA was produced when cells were grown on dextrose or raffinose; transcription was induced by a shift to 2% galactose, and resulted in identical levels of mRNA in wild-type and xpo1-1 cells after either 15 or 60 min in galactose, with or without the high-copy DBP5 plasmid (data not shown). Taken together, these observations suggest that either Xpo1p does not play an essential direct role in mRNA export or high-level expression of Dbp5p restores an mRNA export function of Xpo1-1p without suppressing its protein export defect. Figure 6.(A) Overexpression of Dbp5p prevents xpo1-1 cells from developing an mRNA export defect. In situ hybridization (A, C, E, G) was performed to detect poly(A)+ mRNA in xpo1-1 cells carrying either an empty 2μ vector (A-D), or the same vector expressing Dbp5p (E-H). Cells were stained with DAPI to indicate the location of the nucleus. (B) Localization of Dbp5p expressed from a high-copy plasmid. Indirect immunofluorescence was performed on xpo1-1 cells overexpressing Dbp5p, or transformed with a control 2μ vector, at both 23 and 37°C. Download figure Download PowerPoint Screen for high-copy suppressor of the rat8-2 allele of DBP5 To identify proteins that might interact with Dbp5p, we conducted a screen for high-copy suppressors of the rat8-2 allele. Our previous studies indicated that cells carrying the rat8-2 allele are only moderately temperature sensitive. On a CEN plasmid, rat8-2 supports growth at 34°C and below, but not at 37°C (Snay-Hodge et al., 1998) while this allele is temperature sensitive above 30°C when integrated in place of DBP5. We screened for high-copy suppressors at 34°C in cells carrying an integrated rat8-2 allele (see Materials and methods). Open reading frame (ORF) YMR255w was identified many times in this screen. It is able to suppress the growth defect of the rat8-2 and rss1-37 strains at 34°C but not at higher temperatures (Figure 7A). The 21.5 kDa protein encoded by YMR255w is non-essential, contains a putative coiled-coil domain, and is very basic (pI 9.95) (Nobrega et al., 1998). By two-hybrid analysis, this protein interacts strongly with Gle1p and Rip1p, and less strongly with Dbp5p (Table I). We saw no interaction between Gfd1p and either N-terminal truncation of Dbp5p. V5 epitope-tagged Gfd1p was localized to the cytoplasm and the nuclear rim (Figure 7B). This is a pattern similar to that of Dbp5p, though the nuclear rim staining is less distinct for Gfd1p-V5. Details on the isolation and further characterization of this suppressor will be described in a separate paper (H.V.Colot, P.Stafford, C.A.Hodge and C.N.Cole, in preparation). We have named this gene GFD1 (good for Dbp5p). The same protein has been identified by another laboratory as a suppressor of a GLE1 mutation (Strahm et al., 1999). Figure 7.GFD1 is a high-copy suppressor of rat8-2 and rss1-37. (A) Growth of CSY550 (with the rat8-2 allele integrated at the DBP5 locus) and SPY23(with the rss1-37 allele integrated at the GLE1 locus), with and without a 2μ plasmid containing YMR255w/GFD1. (B) Localization of V5-tagged Gfd1p and V5-tagged Dbp5p in wild-type cells by indirect immunofluorescence using an anti-V5 antibody. Download figure Download PowerPoint Discussion Dbp5p is a shuttling nuclear transport factor Many proteins that participate in nuclear transport shuttle across the nuclear envelope. This includes Gsp1p, all known transport receptors and some of the soluble factors required for mRNA export (e.g. Mex67p, Mtr2p). In this paper, we demonstrate that Dbp5p is a shuttling transport factor, based on its reversible nuclear accumulation in xpo1-1 cells. Overexpression of Dbp5p completely prevented accumulation of poly(A)+ RNA in xpo1-1 nuclei (Figure 6) under conditions where transcription of a galactose-regulated reporter gene was unchanged. At least two possible scenarios can expla