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• W2005855998 abstract "Article1 June 1997free access Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores Alexandra Segref Alexandra Segref University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Kishore Sharma Kishore Sharma EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Valérie Doye Valérie Doye Institut Curie, Section de recherche, CNRS UMR144, 2 rue Lhomond, F-75231 Paris Cedex 05, France Search for more papers by this author Andrea Hellwig Andrea Hellwig University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Jochen Huber Jochen Huber Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopf-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Reinhard Lührmann Reinhard Lührmann Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopf-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Ed Hurt Ed Hurt University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Alexandra Segref Alexandra Segref University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Kishore Sharma Kishore Sharma EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Valérie Doye Valérie Doye Institut Curie, Section de recherche, CNRS UMR144, 2 rue Lhomond, F-75231 Paris Cedex 05, France Search for more papers by this author Andrea Hellwig Andrea Hellwig University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Jochen Huber Jochen Huber Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopf-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Reinhard Lührmann Reinhard Lührmann Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopf-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Ed Hurt Ed Hurt University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Alexandra Segref1, Kishore Sharma2, Valérie Doye3, Andrea Hellwig1, Jochen Huber4, Reinhard Lührmann4 and Ed Hurt1 1University of Heidelberg, BZH, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany 2EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 3Institut Curie, Section de recherche, CNRS UMR144, 2 rue Lhomond, F-75231 Paris Cedex 05, France 4Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopf-Strasse 2, D-35037 Marburg, Germany The EMBO Journal (1997)16:3256-3271https://doi.org/10.1093/emboj/16.11.3256 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5–GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm. Introduction The translocation of molecules across the nuclear membrane occurs through the nuclear pore complexes (Fabre and Hurt, 1994; Görlich and Mattaj, 1996). Different transport routes were identified in the past and a picture is emerging that different transport cargoes use different transport vehicles (Gerace, 1995). For nuclear protein import, the nuclear localization sequence (NLS)–receptor complex consisting of importin/karyopherin α and β subunits binds to the NLS of karyophilic proteins in the cytoplasm (Görlich and Mattaj, 1996), followed by a docking step at the nuclear pore complexes, most likely at FXFG or GLFG repeat sequence containing nucleoporins (Rexach and Blobel, 1995). The translocation of the nuclear protein through the nuclear pore channel is then initiated by GTP hydrolysis, mediated by the small GTPase Ran/TC4 (Moore and Blobel, 1993). RNA export from the nucleus into the cytoplasm is also a signal- and receptor-mediated process (Görlich and Mattaj, 1996). Some of the players participating in the molecular events during RNA export have been recently identified; however, the underlying mechanisms of RNA export remain largely unknown. A cap-binding complex (CBP) which recognizes the monomethylated 5′-end of snRNAs and mRNAs has been shown to mediate U snRNA nuclear export (Izaurralde et al., 1995). Similar to the NLS found in karyophilic proteins, nuclear export sequences (NESs) were recently found in several proteins which shuttle between the nucleus and the cytoplasm (Gerace, 1995). In most cases, these NES-containing proteins are RNA-binding proteins and thus could be involved in the nuclear export of their cognate cargo RNA. This was most convincingly shown for the HIV Rev protein which associates with viral mRNA (Fischer et al., 1994; Bogerd et al., 1995; Stutz et al., 1995) and the hnRNP A1 protein which is bound to mRNA (Michael et al., 1995). HIV Rev transports unspliced and partially spliced viral mRNA from the nucleus into the cytoplasm, and it is the NES of Rev which retargets viral mRNA directly to a cellular export pathway bypassing the splicing machinery (Fischer et al., 1995). Whereas the NES in HIV Rev, PKI (protein kinase inhibitor) and TFIIIA is short (∼10 amino acids in length) and leucine-rich with a typical spacing of the hydrophobic residues (Gerace, 1995), the NES of hnRNP A1 (called M9) is significantly longer and does not conform to this consensus (Michael et al., 1995). Interestingly, the M9 sequence also exhibits NLS activity and was recently shown to interact with a novel import receptor called transportin, which is an importin/karyopherin β homologue (Pollard et al., 1996). Since NESs can induce rapid export of attached passenger proteins (Fischer et al., 1995), a search was undertaken for cellular receptors interacting with the nuclear export signal. By using the yeast two-hybrid system, two related proteins from human and yeast (called hRip and Rip1p, respectively), were identified which specifically interact with the HIV Rev NES (Bogerd et al., 1995; Fritz et al., 1995; Stutz et al., 1995). Interestingly, these Rip proteins share similarity to FG repeat-containing nucleoporins which are also candidates for NES receptors that could facilitate transport of Rev-associated RNPs through the NPC (Stutz et al., 1996). Interestingly, yeast Rip1p is not essential for cell growth, suggesting that additional NES receptors must exist in the cell which are essentially involved in RNA export reactions or have overlapping function. Recently, an essential RNA-export mediator called Gle1p which contains a nuclear export signal and interacts with Rip1p and Nup100p has been identified in yeast (Murphy and Wente, 1996). Gle1p is identical to Rss1p (Del Priore et al., 1996) and Brr3p (Noble and Guthrie, 1996) which have been recently found in other genetic screens. A complementary approach has been undertaken in the yeast Saccharomyces cerevisiae, exploiting its powerful genetics, to dissect the nucleocytoplasmic transport machinery (for review, see Doye and Hurt, 1995). In a genetic screen, a collection of temperature-sensitive mutants was analysed by in situ hybridization for nuclear accumulation of poly(A)+ RNA. Many of the obtained mutants, called rat (mRNA trafficking) and mtr (mRNA transport) mutants (Amberg et al., 1992; Kadowaki et al., 1992), were blocked in nuclear mRNA export. Among the cloned RAT and MTR genes, some of them encode nuclear pore proteins (Doye et al., 1994; Heath et al., 1995; Li et al., 1995). In particular, RAT7/NUP159 (Gorsch et al., 1995) and MTR2 (Kadowaki et al., 1994) are promising candidates for a direct involvement in mRNA export reactions, since thermosensitive mutants show an intranuclear mRNA accumulation shortly after shift to the restrictive temperature. The yeast homologue of RCC1 (the guanine dinucleotide exchanger of Ran) has also been found in such a genetic screen, suggesting that Ran is involved in nuclear RNA export (Kadowaki et al., 1993); similarly, mammalian RCC1 was shown to be required for nuclear RNA export (Cheng et al., 1995). Furthermore, hnRNP proteins in yeast such as Npl3p, which shuttle between the nucleus and the cytoplasm, were also suggested to mediate mRNA export (Lee et al., 1996). Genetic screens in yeast based on synthetic lethality have proven to be extremely useful in identifying a vast number of nuclear pore complex proteins with roles in nuclear pore biogenesis, nuclear pore structure and/or nucleocytoplasmic transport (Doye and Hurt, 1995). Among the many nucleoporins identified so far, some appear to have distinct roles in nuclear protein import, mRNA export and tRNA export, respectively (Simos et al., 1996). Other nucleoporin mutants not only have defects in nucleocytoplasmic transport reactions, but possess exhibit structural abnormalities of the nuclear envelope and NPCs (Doye and Hurt, 1995), making it difficult to assign the primary defect. Furthermore, in many of these nucleoporin mutants the manifestation of transport defects was not immediate after shifting cells to the restrictive condition, making it likely that observed transport defects could be pleiotropic. Recently, a novel nucleoporin complex consisting of Nup120p, Nup85p, Nup84p, band IV, Sec13p and a Sec13 homologue (Seh1p) was identified in yeast and plays roles in coordinated nuclear membrane/NPC biogenesis and in nuclear export of mRNA and tRNA (Siniossoglou et al., 1996). We used a mutant of one member of this complex, Nup85p, which exhibits a poly(A)+ RNA export defect, to search for novel factors involved in nuclear mRNA export. Here, we report the identification of such a component, called Mex67p, which is essential for mRNA transport out of the nucleus. Our studies indicate that Mex67p is likely to be involved in nuclear mRNA export mechanisms. Results A synthetic lethal screen with a mutant allele of NUP85 identifies the essential mRNA export factor Mex67p A nucleoporin complex of six proteins which includes Nup85p is required for nuclear pore biogenesis and RNA export (Siniossoglou et al., 1996). Interestingly, a nup85 null mutant is impaired in both NPC organization and RNA export, whereas cells which express an amino-terminally truncated Nup85p (nup85Δ) and are thermosensitive for growth at 37°C are mainly defective in poly(A)+ RNA export (Siniossoglou et al., 1996). This observation prompted us to perform a synthetic lethal (sl) screen with the nup85Δ allele to identify novel components of the mRNA export machinery (see Materials and methods). In total, 13 sl mutants were isolated which are synthetically lethal with nup85Δ, but not with NUP85. The wild-type gene of a NUP85 interacting component was cloned by complementation of one of these sl mutants (sl102) with a yeast genomic library. The complementing activity was restricted to an uncharacterized yeast gene on chromosome XVI, which encodes a putative protein of 599 amino acids (Figure 1A). The deduced molecular weight of this novel protein is 67.351 kDa. Since this protein plays an essential role in mRNA export (see also later), it was named Mex67p and its gene MEX67 (for Messenger RNA EXport factor of 67 kDa molecular weight). A search in protein sequence data libraries for putative higher eukaryotic homologues of yeast Mex67p (accession No. Z73525) revealed three sequences with significant homology and a similar domain organization (Figure 1A and B); one is a human protein called TAP (accession No. D42085) and the other two are uncharacterized ORFs from C.elegans (accession Nos: C15H11.e/e275614; C15H11.d/e275615). Figure 1.Structural organization and homology of Mex67p. (A) Domain organization and primary amino acid sequence of Mex67p and a homologous human protein called TAP (accession No. U80073). The various domains are highlighted with different colours including a Leucine-Rich Repeat (LRR)-domain (red), an uncharged proline/glutamine/glycine-rich sequence (orange) and the carboxy-terminal domain (blue). The LRR domain was aligned in a way that the four LRRs become evident. Conserved residues in the LRRs are at position 2, 5, 7, 10 and 12 and shown in black. LRRs occur in several other proteins including the ribonuclease inhibitor whose crystal structure has been recently solved. It was speculated that the LRR is involved in protein–protein interactions (Kobe and Deisenhofer, 1994). Within the Mex67p carboxy-terminal domain, a short sequence is underlined, which resembles the HIV Rev NES. The point mutation H(400) to Y in ts mex67-5 is indicated by a star. (B) Multiple sequence alignment of Mex67p with human TAP (accession No. U80073), and two C.elegans ORFs (C15H11.e/e275614; C15H11.d/e275615) using ClustalW1.6 (BCM Search Launcher). Download figure Download PowerPoint Disruption of the MEX67 gene showed that it is essential for cell growth (data not shown). In order to study the in vivo role of Mex67p, thermosensitive mutants were generated by random mutagenesis of the isolated MEX67 gene. Among the three thermosensitive mutants obtained, ts mex67-5 was chosen for further analysis, because it showed no apparent growth defect at 30°C, but completely stopped cell growth shortly after shifting the cells to 37°C (Figure 2A and B). This suggests that an essential cellular process is tightly controlled by Mex67p. Interestingly, ts mex67-5 cells only arrest, but do not die at 37°C, which can be observed by the reversibility of the ts phenotype. Even after prolonged incubation of ts mex67-5 cells at 37°C (e.g. 12 h), >50% of the cells are viable and can regrow if brought back to 30°C (Figure 2C). Thus, mex67-5 is a thermoreversible ts allele. The mutation causing the ts phenotype in mex67-5 is due to a single amino acid exchange, His400 to Tyr400 (Figure 1A). Figure 2.A reversible temperature-sensitive mutant of Mex67p. (A) Growth of mutant mex67-5 and wild-type MEX67 cells at different temperatures. Precultures were diluted in liquid YPD medium and equivalent amounts of cells (undiluted or 1/10 diluted) were spotted onto YPD plates. Plates were incubated for 3 days at 23°C and 30°C, and for 3 days at 37°C. (B) Growth curves of wild-type MEX67 and temperature-sensitive mex67-5 strains in YPD liquid medium at 30°C or 37°C. Cell growth was followed by measuring the optical density at 600 nm (OD600). (C) Cell viability of mex67-5 cells incubated at the restrictive condition. For each time point, the same number of mex67-5 cells (∼1000 per plate) were plated on a YPD-plate. Plates were incubated at 37°C for the indicated time points, before being brought back to 30°C. The number of colonies which formed after 3 days at 30°C were determined. Download figure Download PowerPoint MEX67 was isolated on the basis of synthetic lethality with the mutated nup85Δ gene. When strain sl102 was transformed with plasmid-borne MEX67 alleles, synthetic lethality was only complemented at 30°C by intact MEX67, but not mex67-5 (data not shown). Furthermore, a haploid yeast strain was constructed in which the nup85Δ and the ts mex67-5 allele were combined, but this strain was not viable when the pURA3–MEX67 plasmid was shuffled out on 5-fluoro-orotic acid (FOA) plates (data not shown). This demonstrates synthetic lethality between the two mutant alleles. When we analysed whether MEX67 is also linked to other members of the Nup85p complex or to other nucleoporins, no synthetic lethality was seen below 30°C between mex67-5 and mutant alleles of nup84, seh1 and pom152 (see Materials and methods). However, the combination of mex67-5 and nup84::HIS caused synthetic lethality at 33°C, a temperature at which the single mutants are still able to grow (data not shown). This genetic analysis thus revealed a strong genetic overlap between MEX67 and NUP85, and a weaker or no genetic interaction with mutant alleles of NUP84, SEH1 and POM152, respectively. Since MEX67 was found in conjunction with the nup85Δ mutant allele which causes poly(A)+ RNA to accumulate inside the nucleus, we tested whether Mex67p on its own participates in mRNA export. By in situ hybridization using a FITC-labelled oligonucleotide poly(dT)50 probe, poly(A)+ RNA was localized in the cytoplasm of mex67-5 cells if grown at the permissive temperature (Figure 3A). After shifting the cells for 15–30 min to 37°C, however, poly(A)+ RNA strongly accumulated inside the nucleus in almost all of the mex67-5 cells (Figure 3A). Strikingly, this intranuclear RNA was concentrated in several discrete spots which varied in number (Figure 3A and B). Concomitantly, the nucleolar marker Nop1p was also changed in its distribution in the ts mex67-5 mutant, becoming clustered in several intranuclear spots (Figure 3B). Disintegration of the nucleolus was frequently observed in nucleoporin mutants including nup85 (Goldstein et al., 1996) and nup120 mutants (Aitchison et al., 1995b; Heath et al., 1995). However, the spots containing Nop1p did not co-localize with the accumulated poly(A)+ RNA (Figure 3B), as opposed to two other mRNA export mutants, in which nucleolar antigens and mRNA co-localize inside the nucleus (Kadowaki et al., 1994). In contrast, the distribution of nuclear pore antigens was normal in mex67-5 cells with no tendency of NPC clustering (see also later). It thus appears that, in ts mex67-5 cells, polyadenylated RNA accumulates in discrete intranuclear foci which are not in close contact with the nuclear pore complexes. To determine whether the mex67-5 mutant exhibits morphological abnormalities of the nuclear envelope at the ultrastructural level, thin-section electron microscopy was performed. In ts mex67-5 cells shifted to 37°C, nuclear envelope and NPC morphology, as well as NPC number appeared normal, as judged from the inspection of electron micrographs (Figure 4); however, numerous electron-dense aggregates were seen in the ts mex67-5 mutant incubated at the restrictive temperature (Figure 4, arrows; see also Discussion). In addition, fragmentation of the nucleolus, which can be easily identified because of its typical fibrillar-like appearance, was also visible by electron microscopy (Figure 4, arrowheads). Figure 3.Analysis of nuclear RNA export of nuclear protein import in mex67-5 cells. (A) Accumulation of polyadenylated RNA in the nucleus of ts mex67 cells. Subcellular localization of poly(A)+ RNA was analysed by in situ hybridization with a FITC-labelled oligonucleotide poly(dT) probe. Ts mex67-5 cells were either grown at 30°C or shifted for 30 min to 37°C in YPD-medium. Nuclear DNA was stained by Hoechst 33258 and cells were viewed by Nomarski optics. (B) The fragmented nucleolus does not co-localize with accumulated polyadenylated RNA in ts mex67-5 cells. Thermosensitive mex67 cells were shifted for 30 min to 37°C before cells were fixed and processed for both, in situ hybridization with oligo(dT)-FITC [poly(A)+ RNA] and indirect immunofluorescence using anti-Nop1p antibodies (Nop1p). Both pictures which were obtained from the confocal microscope were merged [poly(A)+ RNA + Nop1p], indicating that poly(A)+ and Nop1p-clusters generally do not co-localize. (C) Analysis of nuclear protein import in ts mex67-5 cells. Intracellular location of the NLS–GFP reporter protein in ts mex67 and MEX67 cells as revealed by fluorescence microscopy. Cells were preincubated for 1 h at 37°C before the in vivo nuclear import analysis was performed essentially after Shulga et al. (1996). − energy: indicates that cells were treated with azide and deoxyglucose. + energy (10′) and + energy (20′), cells after recovery from the drug treatment in glucose-containing medium for 10 and 20 min, respectively. Note that nuclear re-import of GFP–NLS which leaked out into the cytoplasm in energy-depleted cells, is almost complete after 10 min of re-energization, both in mex67-5 and MEX67+ cells. Download figure Download PowerPoint Figure 4.Electron microscopic analysis of ts mex67-5 cells. Wild-type MEX67 and ts mex67-5 cells were grown to the early logarithmic phase at 30°C or 37°C before processing them for thin-section electron microscopic analysis as described under Materials and methods. (a) Wild-type MEX67 cells grown for 2 h at 37°C; (b) ts mex67-5 cells, grown at 30°C; (c and d) ts mex67-5 cells grown for 2 h at 37°C. Small arrows point to nuclear pores, large arrows to electron-dense intranuclear aggregates, which most likely are hRNP clusters, and arrowheads to the fragmented nucleolus. Bar, 0.2 μm. Download figure Download PowerPoint It was further tested whether the biogenesis of other RNA species is altered in ts mex67-5 cells. When rRNA processing was analysed by Northern analysis (Tollervey et al., 1993), no significant impairment of pre-rRNA processing was seen in ts mex67-5 cells after a 30 min shift to 37°C; however after prolonged incubation (e.g. 1 h) at 37°C, a decrease of the 32S and 27SA2 pre-rRNA species, and the simultaneous appearance of the 23S precursor, was noticed. Processing of 20S to mature 18S rRNA, however, was not affected (data not shown). Accordingly, pre-rRNA processing occurs at 37°C in the mex67-5 mutant, but a delay in the cleavage at site A0, A1 and A2 can be measured at later time points of restrictive incubation. Processing and transport of tRNA was normal, since no defect in tRNA splicing and no loss of suppressor tRNA activity was observed in the mex67-5 mutant (data not shown). Finally, mRNA splicing was also not inhibited in the mex67-5 mutant when shifted to 37°C, as seen by the fact that no intron-containing actin mRNA was found (data not shown). To determine whether nuclear protein import is inhibited in ts mex67-5 cells, we tested the nuclear import of the karyophilic reporter protein Matα2–lacZ (Nehrbass et al., 1993); however, no cytoplasmic accumulation was found at the restrictive temperature (data not shown). Since poly(A)+ RNA export is efficiently inhibited in ts mex67-5 cells, one can not exclude that the nuclear reporter protein is no longer synthesized at the restrictive temperature due to the cytoplasmic depletion of reporter mRNA. Therefore, we used a recently developed assay for in vivo nuclear protein import which does not depend on ongoing mRNA synthesis and export (Shulga et al., 1996). In this assay, cells which express and accumulate a NLS–GFP reporter protein inside the nucleus are first poisoned with inhibitors of energy metabolism. This causes leakage of NLS–GFP into the cytoplasm. After washing and resuspending the cells in glucose-containing medium, normal ATP levels are restored, allowing rapid nuclear re-import of NLS–GFP. When the ts mex67-5 mutant expressing NLS–GFP was shifted for 1 h to 37°C and then analysed according to this assay, no defect in nuclear re-import of the NLS–GFP reporter protein was seen as compared with wild-type cells (Figure 3C). In summary, the early onset of an mRNA export defect in ts mex67-5 mutant cells with no apparent impairment in NLS-mediated nuclear protein import suggests that Mex67p is directly involved in nuclear mRNA export reactions. Mex67p is a nuclear pore-associated protein which accumulates in the cytoplasm in ts mex67 cells The functional interaction of Mex67p with nucleoporins and its essential role in nuclear mRNA export suggests that the protein might be localized at the nuclear pores. Therefore, Mex67p was tagged at its carboxy-terminal end with different epitopes derived from Protein A or the Green Fluorescent Protein (see Materials and methods). These fusion proteins were functional since they could complement the lethal phenotype of the mex67 null mutant if expressed from yeast single-copy plasmids (data not shown). Mex67p–ProtA showed a punctuate nuclear envelope staining in wild-type cells and co-clustering with nuclear pore antigens in nup133− cells (data not shown). Also, the in vivo location of GFP-tagged Mex67p was predominantly at the nuclear pores, as seen by the ring-like and punctuate staining of the nuclear envelope (Figure 5A). This staining closely resembles the in vivo labelling seen with a bona fide nucleoporin, NUP49–GFP (Belgareh and Doye, 1997). Finally, the fluorescence signals from Mex67–GFP and Nsp1p largely overlapped, as revealed by double immunofluorescence microscopy (data not shown). Thus, under steady-state conditions, Mex67p shows a preferential location at the nuclear pores. Figure 5.Localization of GFP-tagged Mex67p in living cells. (A) Confocal fluorescence microscopy of living MEX67–GFP cells. Two successive sections, each 0.5 μm thick, are shown for MEX67–GFP, and for comparison, NUP49–GFP cells. The insert shows a higher magnification of MEX67–GFP cells which reveals a typical nuclear pore labelling, with little staining in the nucleoplasm and cytoplasm, and no staining inside the vacuole. (B) Confocal fluorescence microscopy of thermosensitive mex67-5–GFP cells. Living cells growing for the indicated time periods and temperatures in liquid medium were inspected in the confocal fluorescence microscope. The same mex67-5–GFP cells which were immobilized on the microscopic slide, were viewed and photographed twice, once after 10 min shift to 37°C and a second time after a 20 min reshift to 23°C. For control reasons, also MEX67–GFP cells were grown for 15 min at 37°C. Download figure Download PowerPoint A different intracellular location was seen when the mutant protein mex67-5p tagged with GFP was analysed by fluorescence microscopy; mex67-5–GFP complemented the lethal phenotype of the mex67 null disruption mutant at permissive temperatures, but cells were thermosensitive for growth at 37°C (data not shown). When cells were grown at 23°C, mex67-5–GFP was found predominantly at the nuclear envelope; however, this location changed when cells were shifted for as little as 5–10 min to 37°C (Figure 5B). The mex67-5–GFP fusion protein was no longer associated with the nuclear envelope, but detached from the nuclear pores and appeared in many dot-like structures scattered throughout the cytoplasm. When the mutant was reshifted to 23°C, the cytoplasmic clusters disappeared and the nuclear envelope staining often resumed. Wild-type Mex67–GFP did not show this behaviour and was always found at the NPCs, even at 37°C (Figure 5B). To determine whether the cytoplasmic localization of ts mex67-5–GFP seen at 37°C is due to a general dissociation of nucleoporins from the nuclear pores, double indirect immunofluorescence of fixed and spheroplasted ts mex67-5–GFP cells was performed using anti-nucleoporin antibodies. Thermosensitive mex67-5–GFP accumulated in dot-like cytoplasmic structures, whereas nucleoporins recognized by the monoclonal antibody Mab414 remained associated with the NPCs (data not shown). We next tested whether ProtA–Nup85p becomes mislocalized in ts mex67-5 cells shifted for 1 h to 37°C, but again this nucleoporin remained exclusively bound at the NPCs. Finally, Mex67p–GFP was strictly found at the nuclear pores in nup85Δ mutant cells, shifted for 2 h to the restrictive temperature (data not shown). Taken together, these data show that mutated mex67-5–GFP, but not other nucleoporins, dissociates from the nuclear pores into the cytoplasm under restrictive conditions and is retargeted to the nuclear envelope when shifted to permissive temperature. A short sequence in the carboxy-terminal domain is essential for in vivo function of Mex67p The sequence 549LELLNKLHL557 in the carboxy-terminal domain of Mex67p resembles the NES of HIV Rev (Figure 6A; see also Figure 1A). We therefore coupled this NES-resembling peptide (CLELLNKLHL) and a mutant form (CLELPNKLHL; see also later) to 125I-labelled bovine serum albumin (BSA) and microinjected the conjugates into Xenopus oocyte nuclei. The NES-mediated export was then followed by: (i) determining the amount of Mex67p NES–BSA in the nucleus and cytoplasm; and (ii) measuring competition of Rev-mediated RNA export in the presence of Mex67p NES–peptides coupled to BSA. The NES-like sequence of Mex67p, but not its mutant form, exhibits a nuclear export activity in the Xenopus oocyte system which is comparable in its efficiency to the activity of the Rev NES (Figure 7A). Furthermore, the Rev-mediated export of pAd46 RNA, harbouring the Rev-responsive element (RRE), and export of U1ΔSm RNA were also competitively inhibited by intact, but not mutated Mex67p NES–BSA (Figure 7B). This showed that the NES-like sequence of Mex67p exhibits a nuclear export activity in the Xenopus oocyte system, but it is uncertain whether this sequence also has NES-activity in the context of the native Mex67p protein (see also Discussion). Figure 6.Muta" @default.
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• W2005855998 date "1997-06-01" @default.
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• W2005855998 title "Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores" @default.
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