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• W2029103676 abstract "Article1 August 2001free access An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport Anne de Bruyn Kops Anne de Bruyn Kops Department of Biochemistry and Biophysics, UCSF Medical School, 513 Parnassus Avenue, San Francisco, CA, 94143 USA Search for more papers by this author Christine Guthrie Corresponding Author Christine Guthrie Department of Biochemistry and Biophysics, UCSF Medical School, 513 Parnassus Avenue, San Francisco, CA, 94143 USA Search for more papers by this author Anne de Bruyn Kops Anne de Bruyn Kops Department of Biochemistry and Biophysics, UCSF Medical School, 513 Parnassus Avenue, San Francisco, CA, 94143 USA Search for more papers by this author Christine Guthrie Corresponding Author Christine Guthrie Department of Biochemistry and Biophysics, UCSF Medical School, 513 Parnassus Avenue, San Francisco, CA, 94143 USA Search for more papers by this author Author Information Anne de Bruyn Kops1 and Christine Guthrie 1 1Department of Biochemistry and Biophysics, UCSF Medical School, 513 Parnassus Avenue, San Francisco, CA, 94143 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4183-4193https://doi.org/10.1093/emboj/20.15.4183 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a Δnup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery. Introduction The presence of a nuclear envelope (NE) in eukaryotes divides the cell into nuclear and cytoplasmic compartments, greatly enhancing its capacity to control gene expression. Transport of proteins and ribonucleoprotein particles (RNPs) between the nucleus and cytoplasm through the nuclear pore complex (NPC) is central to this ability (reviewed in Görlich and Kutay, 1999; Nakielny and Dreyfuss, 1999). The structure of the NPC is conserved from yeast to humans, consisting of a highly symmetrical core structure organized around a central pore channel as well as peripheral cytoplasmic filaments and nucleoplasmic basket structures thought to play key roles in initiation and termination of transport pathways (reviewed in Franke and Scheer, 1974; Davis, 1995; Stoffler et al., 1999; Wente, 2000). Transport of different types of protein and RNP cargoes through the NPC is mediated by specific protein carriers, including members of the karyopherin β family and a number of unrelated transport factors such as Ntf2 and Mex67 (reviewed in Pemberton et al., 1998; Wozniak et al., 1998; Görlich and Kutay, 1999). Interactions between carriers and nucleoporins lining the channel of the pore are thought to be important for transport (reviewed in Fabre and Hurt, 1997; Pemberton et al., 1997; Wozniak et al., 1998; Görlich and Kutay, 1999; Wente, 2000), although the precise roles of particular nucleoporins are not well understood. Our knowledge of the functional architecture of the yeast nuclear pore has benefited greatly from a detailed study of isolated NPCs carried out recently by Rout et al. (2000). The study produced a working model of the NPC, confirming the presence of ∼30 yeast nucleoporins at the pore and placing them spatially with respect to known ultrastructural features such as the central channel and nuclear basket. Many of these proteins are likely to function in the targeting of transport substrates to the NPC and their passage to the cytoplasm. In addition to nucleoporins, the NE around the NPC could also play a key role in transport, possibly providing binding sites for transport factors or anchoring pore structures. Interestingly, an antibody against gp210, a mammalian integral membrane protein located in the pore membrane domain bridging the inner and outer NE membranes (reviewed in Gerace and Burke, 1988; Dingwall and Laskey, 1992; Gant and Wilson, 1997), has been shown to inhibit nuclear import (Greber and Gerace, 1992). Although the mechanism of the inhibition is unknown, the result suggests that integral membrane proteins can affect transport events. In yeast, three integral membrane proteins specific to the NE have been identified. Two of these, Pom152p and Pom34p (Wozniak et al., 1994; Strambio-de-Castillia et al., 1995; Tcheperegine et al., 1999; Rout et al., 2000), co-purify with yeast nuclear pores. Both are believed to reside in the pore membrane domain and to function in nuclear pore assembly and anchoring. The third, Ndc1p, is an inner envelope protein that interacts with both the NPC and the spindle pole body (Chial et al., 1998). In addition, three yeast proteins, Snl1p, Nem1p and Spo7p, common to the NE and the endoplasmic reticulum, have also been shown to affect the NPC (Ho et al., 1998; Siniossoglou et al., 1998). However, none of the known yeast NE integral membrane proteins has been reported to affect nuclear transport. Here we report the characterization of an essential yeast protein, Brr6p, which has the properties of an NE integral membrane protein and affects specific nuclear transport pathways. The BRR6 gene interacts genetically with a subset of nucleoporins, and loss of Brr6p function causes redistribution of Nsp1p and Nup188-green fluorescent protein (GFP) as well as aberrant envelope and pore morphologies. Strikingly, the brr6-1 cold-sensitive (cs) allele accumulates mRNA and a nuclear export signal (NES) protein reporter at the nuclear rim. Thus, Brr6p represents the first example of a yeast NE integral membrane protein that impacts nuclear transport. Results BRR6 was identified through complementation of the growth defect of brr6-412, one of two original isolates of the brr6-1 cs mutant obtained in an in situ hybridization screen for cs mRNA export mutants (see Materials and methods). The 197 aa BRR6 open reading frame (ORF) [Saccharomyces cerevisiae Genome Database (SGD) accession No. YGL247w] is predicted to encode an essential 22.8 kDa protein of unknown function. Disruption of the BRR6 ORF with the HIS3 marker confirmed that the gene is essential. The brr6-1 allele was found to contain a single, conservative arginine to lysine change at amino acid 110. Isogenic BRR6 and brr6-1 strains were generated by integrating wild-type and mutant alleles into a Δbrr6::HIS3 deletion strain (see Materials and methods for details). The resulting brr6-1 mutant showed a moderate growth defect at 30°C, which was exacerbated at 16°C, while the BRR6 strain was indistinguishable from the wild-type parent (data not shown). The brr6-1 mutant accumulates mRNA in the nucleus and at the nuclear periphery Using a digoxygenin-labeled dT50 probe, we examined the mRNA in situ hybridization patterns in BRR6 and brr6-1 cells maintained at 30°C (Figure 1) or shifted to 16°C (data not shown). At both temperatures, BRR6 cells showed the whole cell dT50 staining typical of wild-type cells. In contrast, brr6-1 cells had clear staining in the cell nucleus at 30°C as well as at 16°C. Thus, brr6-1 exhibits a constitutive nuclear mRNA export defect. A strain in which the only copy of BRR6 was under the control of the repressible GAL1-GAL10 promoter also showed both a growth defect and nuclear mRNA accumulation when grown for 5 h in media containing glucose (data not shown), indicating that these are most likely loss-of-function phenotypes. In some brr6-1 cells, dT50 signal was clearly concentrated at the nuclear rim (Figure 1, insert), suggesting that BRR6 may play a role in a step of mRNA export occurring at or near the nuclear pore. Figure 1.The brr6-1 mutant accumulates bulk poly(A) RNA in the nucleus and at the nuclear rim. Shown are the mRNA localization patterns in BRR6 and brr6-1 cells at 30°C determined by in situ hybridization with a digoxygenin-labeled oligo dT50 probe. DAPI staining was used to confirm the locations of the cell nuclei. The inserts in the brr6-1 panels show dT50 and DAPI staining of a single nucleus enlarged to show the mRNA accumulation at the nuclear rim evident in some mutant cells. Bar, 10 μm. Download figure Download PowerPoint brr6-1 is defective in NES protein transport The factors known to affect mRNA export in yeast can be divided into two general categories: those that appear to be dedicated to mRNA and those that also affect protein transport pathways (reviewed in Nakielny and Dreyfuss, 1999). Our in situ hybridization results suggested a role for BRR6 in mRNA export; to assess whether BRR6 also functions in protein transport, we examined the localization of a number of different GFP-tagged protein transport reporters in living BRR6 and brr6-1 cells. Reporters were selected that are known to utilize different protein transport pathways. The set included diffusible and non-diffusible SV40 nuclear localization signal (NLS)-GFP constructs [NLS-GFP, NLS(GFP)3], an SV40 NLS/NES-GFP reporter [NLS/NES(GFP)2], a ribosomal protein NLS reporter (L25-GFP) as well as GFP-tagged forms of two known mRNA binding proteins, Npl3p and Nab2p. Of the reporters tested, only the NLS/NES(GFP)2 construct showed any change in localization. In BRR6 cells (Figure 2), the reporter showed the expected wild-type cytoplasmic distribution reported previously (Stade et al., 1997). Interestingly, about half of the brr6-1 mutant cells with GFP signal showed a pronounced accumulation of the reporter at the nuclear rim, consistent with a defect in NES protein transport. In contrast, the distribution of an NLS(GFP)3 reporter lacking the NES sequence was unaffected in brr6-1 (Figure 2). Similarly, no defects were observed using a diffusible NLS-GFP reporter (data not shown) in either steady state experiments or in the kinetic protein import assay developed by Goldfarb and colleagues (Shulga, 1996). The distributions of the L25-GFP reporter, Npl3p and Nab2p were also unchanged in brr6-1 (data not shown). The effect on NLS/NES(GFP)2 but not other protein reporters implies a defect specific to the NES protein transport pathway. Interestingly, the NES protein exporter Xpo1p (reviewed in Görlich and Kutay, 1999) continued to show wild-type nucleoplasmic and nuclear rim distribution in brr6-1 using an Xpo1-GFP fusion protein (data not shown). Figure 2.The brr6-1 mutation causes a defect in NES protein export. Shown are the localization patterns of NLS/NES(GFP)2 and NLS(GFP)3 reporters in BRR6 and brr6-1 cells as indicated. The NLS/NES construct accumulates at the nuclear rim in brr6-1 as confirmed by DAPI staining (data not shown). The NLS construct shows nucleoplasmic staining in both mutant and wild-type cells. Bar, 10 μm. Download figure Download PowerPoint Brr6p is located at the nuclear rim The mRNA export and NES protein transport defects observed in brr6-1, along with the apparent absence of effects on other protein reporters, argue that BRR6 plays a role in mRNA and NES transport pathways. The accumulation of both mRNA and the NES reporter at the nuclear rim suggests that BRR6 may act at the NE. To determine the location of the BRR6 gene product, we examined the distribution of a Brr6-GFP fusion protein in living cells by fluorescence microscopy. When Brr6-GFP was expressed from a low copy plasmid, the GFP signal was observed predominantly at the nuclear rim, suggesting that Brr6p associates with the NE (Figure 3A). Notably, the Brr6-GFP rim staining pattern was punctate, similar to that seen with nuclear pore components (Rout et al., 2000). The pattern was distinctly different from the uniform rim distribution of non-pore-associated NE integral membrane proteins, such as Spo7p and Nem1p in yeast (Siniossoglou et al., 1998), suggesting that Brr6p may be located at or near the pores. Interestingly, overexpression of Brr6-GFP resulted in dramatic localization to both the nuclear and cellular peripheries (Figure 3A). The latter distribution resembles that of the ER markers Sec63-GFP and GFP-HDEL in living yeast cells (Prinz et al., 2000) and most likely represents accumulation of excess Brr6p in the peripheral ER. Similar localization in both the NE and the ER has been shown for Spo7p and Nem1p (Siniossoglou et al., 1998) and for the mammalian nuclear pore membrane protein, Pom121, upon overexpression (Soderqvist et al., 1997). Figure 3.Brr6-GFP localizes to the nuclear rim. (A) The localization patterns of a Brr6-GFP fusion protein expressed from a low copy (CEN/ARS) vector or a high copy 2 μm plasmid as indicated. At low copy, Brr6-GFP shows predominantly punctate nuclear rim staining. Upon overexpression, Brr6-GFP accumulates at the nuclear and cell peripheries. (B) The effect of BRR6 (BRR6/pRS424) overexpression relative to the empty vector (pRS424) on growth of wild-type (W303), Δnup116::HIS3, Δnup1::HIS3 and Δnup188::HIS3 strains (all W303 strain background) on -TRP medium. BRR6 overexpression causes dramatic growth defects in Δnup1 and Δnup188 cells with only mild effects on wild-type and Δnup116 cells. (C) Nup188-GFP and Brr6-GFP (Brr6-GFP/pRS424) localization patterns in wild-type (RS456) and Δnup133 cells. In contrast to Nup188-GFP, Brr6-GFP fails to show clustering in the Δnup133 mutant. Bar, 10 μm. Download figure Download PowerPoint BRR6 interacts genetically with nucleoporin genes Consistent with the possibility that Brr6p is located at or near the NPC, BRR6 shows genetic interactions with nuclear pore components. A haploid double mutant carrying a cs allele of the nucleoporin, nup188 (brr7-1; A.de Bruyn Kops and C.Guthrie, unpublished results), and the Δbrr6::HIS3 deletion covered by brr6-1 on a TRP plasmid and wild-type BRR6 on a URA plasmid are viable, whereas loss of the URA plasmid on 5′-fluoroorotic acid (5-FOA)-containing medium is lethal (data not shown), indicating that brr6-1 is synthetically lethal with nup188cs/brr7-1. Moreover, overexpression of BRR6 (BRR6 2 μm plasmid) in Δnup188 and Δnup1 deletion strains dramatically impaired growth, while overexpression had only a modest effect in wild-type W303 or Δnup116 cells (Figure 3B). A Δnup2 strain and a C-terminal Δnic96 deletion mutant [Δnic96(532–839)] as well as the xpo1-1 and gle2-1 (RNA export factor) temperature-sensitive (ts) mutants also showed synthetic lethality with BRR6 using this assay (data not shown). Interestingly, the locations of Nup1p (Rout et al., 2000) and a portion of Nic96p (Fahrenkrog et al., 1998) detected by immuno-electron microscopy coincide with the nuclear basket, pointing to a possible impact of BRR6 on basket function. Importantly, a number of strains tested containing deletions or mutations in other nucleoporins [including Δnup100, Δnup133, Δnup145, Δnic96(28–63), and brr3/gle1cs and ts alleles] showed no effect, indicating that the observed interaction is specific to a subset of nucleoporins and transport components. Brr6-GFP localization is unchanged in a pore-clustering mutant Although BRR6 interacts genetically with nucleoporins, Brr6p was not identified in the recent NPC preparations reported by Rout et al. (2000), raising the possibility that Brr6p may interact with the pore without being part of the NPC per se. Another criterion that is frequently used to evaluate if a protein is a nucleoporin is whether its distribution is altered in one of several nucleoporin mutants (e.g. Δnup133; Doye et al., 1994; Pemberton et al., 1995) known to cause pore clustering. To test the behavior of Brr6p in this assay, we compared the distribution of Brr6-GFP and a tagged nucleoporin, Nup188-GFP, in a Δnup133 strain. As predicted, the pattern of Nup188-GFP signal changed from nuclear rim in the wild-type strain to a characteristic single bright nuclear spot in the Δnup133 mutant (Figure 3C). Attempts to localize Brr6-GFP in the Δnup133 and other nucleoporin deletion mutants known to cause pore clustering using a low copy Brr6-GFP construct were unsuccessful because the GFP signal was very low. Efforts to localize the protein by immunofluorescence using a number of epitope tags have also been unsuccessful, most likely because of the small size, membrane association and apparent low abundance of the protein. Similarly, we have not yet been able to generate a usable antibody against Brr6p itself. However, when the experiment was performed using a high copy Brr6-GFP construct (Figure 3C), wild-type and Δnup133 cells showed indistinguishable Brr6-GFP distributions. A caveat to these experiments is the possibility that excess Brr6-GFP might remain distributed throughout the NE. Nonetheless, given the dramatic nature of the clustering phenotype, we would still expect to see intensified signal in one location if clustering occurred. The failure of Brr6-GFP to cluster in this assay distinguishes Brr6p from nucleoporins as well as from the pore membrane proteins, Pom152p and Pom34p, which behave like canonical nucleoporins in both this assay and in the NPC preparation (Rout et al., 2000). The extraction profile of Brr6p from crude lysates typifies an integral membrane protein The Brr6-GFP localization results indicate that Brr6p is most likely a membrane component concentrated at the NE. As such, Brr6p could be either a peripheral membrane protein, associated with the envelope via interactions with nucleoporins or other envelope-associated proteins, or an integral membrane protein. One of the characteristics that distinguishes integral from peripheral membrane proteins is their relative solubility under different extraction conditions. While peripheral proteins can be readily removed by treatment with high ionic strength or high pH, solubilization of integral membrane proteins requires detergent to disrupt lipid–lipid and lipid–protein interactions. We thus tested the ability of high salt, high pH and detergent to solubilize Brr6p. Whole-cell extracts were prepared from Δbrr6 cells containing a low copy BRR6-GFP construct using four different extraction conditions (see Materials and methods). Extracts were centrifuged at 100 000 g and pellet and supernatant samples were assayed by western blotting with an antibody specific for GFP. Brr6-GFP remained associated with the cell pellets in samples extracted under low salt, high salt or high pH conditions but was efficiently extracted in low salt buffer containing 4% Triton X-100 (Figure 4A). This is the profile expected for an integral membrane protein and is similar to that observed for the known nuclear envelope integral membrane protein Pom152p (Figure 4B). In contrast, HA-tagged Nup188p, a non-integral membrane nuclear pore component, was extracted under both high salt and high pH conditions (Figure 4C). Thus, in crude lysates, Brr6p exhibits properties characteristic of integral membrane proteins. Figure 4.The extraction profile of Brr6p from crude lysates typifies an integral membrane protein. Shown are analyses of Brr6-GFP (A), Pom152 (B) and Nup188-HA (C) extracted from whole-cell lysates using four different extraction conditions: (i) 150 mM NaCl; (ii) 1 M NaCl; (iii) 150 mM NaCl, 100 mM sodium carbonate; (iv) 150 mM NaCl, 4% Triton X-100. Supernatant (S) and pellet (P) lanes are indicated. Western blots were probed with antibodies against GFP, Pom152 or the HA tag. Brr6-GFP and Pom152 are present in the supernatant only after detergent treatment, while Nup188-HA is also evident in high salt and high pH supernatants. Download figure Download PowerPoint The C-terminus of Brr6p is sequestered in the NE lumen Consistent with the solubility behavior of Brr6p in extracts, sequence analysis of the BRR6 ORF revealed two hydrophobic stretches with the potential to function as transmembrane domains. One is located near the C-terminus (residues 158–174) and a second, somewhat weaker candidate, is present near the N-terminus (residues 58–74) (Figure 5A). The use of one or both of these sequences to anchor Brr6 in the membrane would dictate the topology of the protein with respect to the bilayer and hence, the availability of different parts of the protein for functional interactions. We were particularly interested in determining the orientation of the brr6-1 mutation (aa 110) with respect to the NE lumen. The mutation is located between the two predicted transmembrane sequences and lies within a region of BRR6 that shows striking homology (31% identical, 49% similar) to a Saccharomyces cerevisiae karyopherin gene, KAP123 (residues 247–312), implicated in ribosomal protein import and mRNA export (Rout et al., 1997; Schlenstedt et al., 1997; Seedorf and Silver, 1997) (Figure 5A). The region of homology comprises ∼1/3 of the BRR6 ORF (residues 52–120) and could reflect the interaction of Brr6p and Kap123p with a common transport factor or nucleoporin. For both the region of KAP123 homology and the brr6-1 mutation to be accessible to the transport machinery, Brr6p would have to be anchored in the membrane by the C-terminal predicted transmembrane domain, with the C-terminus sequestered in the NE lumen. Figure 5.Trypsin digestion yields a detergent-sensitive C-terminal Brr6 fragment. (A) The BRR6 ORF, showing the locations of the brr6-1 mutation (asterisk), the KAP123 homology and the two predicted transmembrane domains. A sequence alignment of the homologous portions of BRR6 and KAP123 is shown. No significant similarity was found between BRR6 and the karyopherin β family prototype, KAP95. (B) The results of trypsin digestion experiments carried out on whole-cell lysates containing Brr6-GFP and polyoma-Brr6 constructs tagged at the C- and N-termini, respectively. Western blot analyses using antibodies against the GFP or polyoma tags demonstrate the protection of a C-terminal Brr6-GFP fragment (asterisk), which is sensitive to detergent. No protected N-terminal fragments are observed. (C) The likely topology of Brr6p in the membrane. The ends of the region of KAP123 homology (arrows) and the site of the brr6-1 mutation (asterisk) are indicated. Download figure Download PowerPoint To investigate the membrane topology of Brr6p, we used trypsin digestion to determine whether the N- and/or C-termini of the protein were protected from proteolysis in the presence and absence of Triton X-100. We carried out trypsin digests on crude lysates containing Brr6p bearing either a C-terminal GFP tag or an N-terminal polyoma tag. The results of a typical experiment are shown in Figure 5B. In the absence of trypsin, a protein of ∼49 kDa corresponding to full-length Brr6-GFP is present in the presence or absence of detergent (lanes 1 and 4). Trypsin digestion in the absence of detergent results in a protected fragment of ∼29 kDa apparent mol. wt (lane 2). The fragment is specific to the trypsin digestion because addition of soybean trypsin inhibitor results in primarily the full-length protein (lane 3). Although it is possible that the trypsin protection results from protein conformation rather than membrane association, the detergent sensitivity (lane 5) argues in favor of protection by the bilayer. The size of the protected Brr6-GFP fragment correlates precisely with the length expected if Brr6p were anchored in the membrane via the C-terminal-most predicted transmembrane domain, with the C-terminus extending into the lumen of the NE. In extracts containing the N-terminally tagged polyoma-Brr6p there were no protected fragments (Figure 5B). A band of ∼26 kDa corresponding to the full-length product was seen in the absence of trypsin or when protease inhibitor was added (lanes 1, 3 and 4). However, no bands were observed when trypsin was included, either in the absence or presence of detergent (lanes 2 and 5). These results are consistent with a topology in which the Brr6 N-terminus, including the site of the brr6-1 mutation and the region of homology with Kap123p, is located outside the lumen (Figure 5C). Such an orientation would make interaction of these regions with the transport machinery feasible. Depletion of Brr6p alters the localization of two nucleoporins Taken together, the BRR6 genetic interactions and the Brr6-GFP localization and extraction data argue that Brr6p is an NE membrane protein located near, and interacting with, the NPC. To further examine the relationship between Brr6p and the NPC, we tested whether the localization of two nucleoporins, Nup188p and Nsp1p, is altered under conditions where BRR6 expression is inhibited. The localization of a GFP-tagged Nup188 protein was examined in living cells in which the sole copy of Brr6p was expressed from its own promoter (BRR6-Myc) or the repressible GAL1-GAL10 promoter (galBRR6-Myc) (see Materials and methods). When these strains were assayed for growth on YEP plates containing galactose or glucose, BRR6-Myc grew well on both carbon sources while galBRR6-Myc grew on galactose but failed to grow on glucose. When cells cultured in YEP media containing galactose were switched to YEP with glucose, a growth defect was observed in the galBRR6-Myc but not in the BRR6-Myc strain by 6 h. When Nup188-GFP localization was examined in living cells, the GFP signal became consolidated into a single spot in most of the galBRR6-Myc cells by 5 h following a switch to glucose-containing medium (Figure 6A). Co-staining with DAPI confirmed that this spot was located at the nuclear rim (data not shown). No change in Nup188-GFP localization was observed in the BRR6-Myc strain. Similar results were obtained when Nsp1p was detected by immunofluorescence microscopy in fixed galBRR6-Myc and BRR6-Myc cells grown in galactose and switched to glucose medium (Figure 6A). When cells were maintained in galactose, no change in Nup188-GFP or Nsp1p localization was observed in BRR6-Myc or the majority of galBRR6-Myc cells (data not shown). A small portion (<10%) of galBRR6-Myc cells showed concentrations of signal at a few spots along the nuclear rim (data not shown), probably reflecting suboptimal expression of Brr6p from the galactose promoter in some cells. In the brr6-1 mutant, Nup188-GFP and Nsp1p appeared to be decreased in nuclear rim signal rather than redistributed to a single spot (Figure 6B). Figure 6.Depletion of Brr6p causes redistribution of nucleoporins. (A) Nup188-GFP and Nsp1p localizations under conditions where Brr6p expressed from the GAL1-GAL10 promoter (galBRR6-Myc) was depleted by glucose inhibition. Nup188-GFP was assayed in living cells and Nsp1p was detected by indirect immunofluorescence in fixed cells. Growth in glucose medium for 5 h resulted in a consolidation of the Nup188-GFP and Nsp1p signals (examples indicated with arrows) in the galBRR6-Myc strain but not in BRR6-Myc in which BRR6 is under the control of its own promoter. (B) Nup188-GFP and Nsp1p distributions in BRR6 and brr6-1 cells. Bar, 10 μm. Download figure Download PowerPoint Brr6p depletion causes changes in NPC distribution and envelope morphology The redistribution of Nup188-GFP and Nsp1p from a punctate rim pattern to a single spot observed upon Brr6p depletion is reminiscent of the pore-clustering phenotype exhibited by Δnup133 (Doye et al., 1994; Pemberton et al., 1995). Clustering is also seen in a mutant of GLE2, an NPC-associated transport factor, as well as in certain nucleoporin mutants (reviewed in Wente, 2000). In the gle2-1 mutant, the phenotype is accompanied by major changes in NE morphology (reviewed in Fabre and Hurt, 1997; Wente, 2000). In other mutants, including Δnup133, pores aggregate but little alteration of the envelope is observed (Doye et al., 1994; Aitchison et al., 1995; Heath et al., 1995; Li et al., 1995). To determine whether changes in pore or membrane ultrastructure underlie the Nup188-GFP and Nsp1p redistribution observed following Brr6p depletion, we carried out electron microscopy on galBRR6-Myc and BRR6-Myc strains grown in galactose and switched to glucose for 5 h. Aberrant pore and envelope morphology consisting of a bulge in the outer NE overlying a large electron-dense mass was observed in >50% of galBRR6-Myc nuclei following a shift to glucose-containing medium (Figure 7A). The abnormal structures appeared both individually and in clusters and strongly resembled the NE ‘herniations’ reported previously for the gle2-1 and Δnup116 mutants (Wente and Blobel, 1993; Murphy et al., 1996; Bailer et al., 1998). As with the Nup188-GFP and Nsp1p localization phenotypes, abnormal membrane morphology was seen in a small portion (<5%) of the galBRR6-Myc cells maintained in galactose-containing medium. In contrast, the pore and envelope morphologies in the BRR6-Myc strain in either medium were indistinguishable from that seen in an untagged wild-type strain, BRR6, grown in glu" @default.
• W2029103676 created "2016-06-24" @default.
• W2029103676 creator A5045054113 @default.
• W2029103676 date "2001-08-01" @default.
• W2029103676 modified "2023-10-16" @default.
• W2029103676 title "An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport" @default.
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• W2029103676 doi "https://doi.org/10.1093/emboj/20.15.4183" @default.
• W2029103676 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/149179" @default.
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