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Article |
Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance
Correspondence to Karsten Weis: kweis{at}berkeley.edu
The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.
Abbreviations used in this paper: AAHD, amphipathic
-helical domain; LB, lysis buffer; LC, liquid chromatography; MBP, maltose-binding protein; MS, mass spectrometry; NE, nuclear envelope; NPC, nuclear pore complex; TAP, tandem affinity purification.
© 2009 Onischenko et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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500 polypeptides, but because of their highly symmetrical organization, NPCs consist of only 30 distinct nucleoporins, all present in 8, 16, or 32 copies per NPC (Rout et al., 2000; Cronshaw et al., 2002; Alber et al., 2007a,b). Despite our detailed knowledge of the protein composition of NPCs, little is known about the pathways that lead to NPC assembly, and it remains poorly understood how NPC biosynthesis is spatially or temporally coordinated. In higher eukaryotes that undergo an open mitosis, two NPC assembly pathways can be distinguished. The first pathway occurs upon completion of mitosis, when nucleoporin subcomplexes and membrane vesicles are recruited to chromatin during NE reformation (Rabut et al., 2004; Antonin et al., 2008; Dultz et al., 2008). In vitro studies using an NE assembly assay in Xenopus laevis egg extracts revealed discrete NPC assembly steps that are initiated by an early recruitment of the Nup107-160 complex (Belgareh et al., 2001; Harel et al., 2003; Walther et al., 2003) to chromatin by the DNA-interacting nucleoporin ELYS (embryonic large molecule derived from yolk sac)/Mel28 (Rasala et al., 2006; Franz et al., 2007; Gillespie et al., 2007). This is followed by the recruitment of membrane vesicles containing the transmembrane nucleoporins POM121 and NDC1, which in turn leads to the incorporation of Nup155 (Nup157/Nup170 in yeast) and Nup53 (Nup53/Nup59 in yeast) and ultimately to the formation of complete and functional NPCs (Antonin et al., 2008). The second pathway occurs during interphase when new NPCs are synthesized and inserted into intact NEs (Maul et al., 1972; Winey et al., 1997; D'Angelo, and Hetzer, 2006). Little is known about the mechanism of NPC biogenesis in interphase, and it is unclear how NPCs are inserted into the two lipid bilayers of an intact NE. De novo NPC synthesis during interphase occurs in all eukaryotes, but many unicellular eukaryotes such as the yeast S. cerevisiae must rely exclusively on NPC insertion into intact double membranes as they undergo a closed mitosis with no NE breakdown. Transmembrane nucleoporins are thought to be crucial for interphase NPC biogenesis by anchoring and recruiting soluble NPC components to the nuclear membrane (Suntharalingam, and Wente, 2003; Antonin et al., 2008). In both mammalian and yeast cells, only three transmembrane nucleoporins have been characterized: POM121, gp210, and NDC1 in mammals and Pom34, Pom152, and Ndc1 in yeast (Wozniak, and Blobel, 1992; Hallberg et al., 1993; Wozniak et al., 1994; Chial et al., 1998; Rout et al., 2000; Mansfeld et al., 2006; Stavru et al., 2006a). In mammalian cells, RNAi-mediated silencing of POM121 alone or in combination with gp210 had no effect on cell viability but caused somewhat variable phenotypes with respect to the distribution of other nucleoporins (Antonin et al., 2005; Stavru et al., 2006b). RNAi depletion of NDC1, the only evolutionarily conserved transmembrane nucleoporin, caused more severe phenotypes and affected NE localization of multiple nucleoporins (Mansfeld et al., 2006; Stavru et al., 2006a). Interestingly, the knockout of NDC1 in C. elegans also affected nucleoporin distribution, but NDC1 is not absolutely essential for the viability of the animals (Stavru et al., 2006a), suggesting that the role of the three transmembrane nucleoporins in NPC biogenesis and maintenance might be redundant. In agreement with this, neither POM152 nor POM34 is essential for cell viability or NPC function in budding yeast. However, additional depletion of Ndc1 caused severe structural abnormalities of NPC and led to the appearance of enlarged pore structures within the NE (Madrid et al., 2006).
To better understand the role of transmembrane nucleoporins in NPC biogenesis, we identified the direct interaction partners of Ndc1 in budding yeast. We show that Ndc1 can be isolated in a subcomplex with Pom152 and Pom34 and that Ndc1 alternatively interacts with either Nup53 or Nup59. In turn, Nup53 and Nup59 directly bind to the core nucleoporins Nup170 and Nup157, thus establishing a connection between the core of NPC and the pore membrane. The removal of Ndc1-interacting partners leads to severe ultrastructural NPC defects resembling the enlarged pore phenotype observed upon depletion of Ndc1 in pom152 deletion mutants (Madrid et al., 2006). Using the photoconvertible fluorescent protein Dendra (Gurskaya et al., 2006), we found that depletion of Pom34 in the absence of Nup53 and Nup59 leads to the reversible mislocalization of newly synthesized Nup82, indicating a block in the assembly of new NPCs. Our results demonstrate critical but redundant roles of the Ndc1-interacting nucleoporins in the assembly and structural integrity of yeast NPCs.
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It was recently shown that Pom152 and Pom34 form a stable, ring-like structure, and it was proposed that this complex is stabilized by interactions involving the large luminal domain of Pom152 (Alber et al., 2007a,b). We therefore decided to test whether the interactions between Ndc1 and the two Poms are dependent on the luminal domain of Pom152. We performed Ndc1 purifications from yeast strains expressing truncated versions of myc-tagged Pom152. A C-terminal Pom152 truncation (Pom1521–301), which lacks most of the luminal domain (Tcheperegine et al., 1999), was efficiently coprecipitated with Ndc1 and still supported the recruitment of Pom34. In contrast, an N-terminal truncation of Pom152 (Pom152170–1,337), essentially lacking the entire cytosolic domain (Tcheperegine et al., 1999), did not copurify with Ndc1 and prevented efficient recruitment of Pom34 (Fig. 1, F and G). Consistent with this, Pom1521–301 localized correctly to NE, whereas Pom152170–1,337 did not (Fig. 1 H). This mislocalization of Pom152170–1,337 was specific, and no effect on Ndc1 or Pom34 localization could be observed in these cells (Fig. S1 A). Of note, Pom152170–1,337 was correctly inserted into the membrane as judged by its presence in the membrane fraction and by its detergent extractability (Tcheperegine et al., 1999; unpublished data).
Together, these findings indicate that all three yeast transmembrane nucleoporins form a distinct NPC subcomplex, which we refer to as the Ndc1 subcomplex. This complex is held together by at least two direct interactions: (1) between Ndc1 and Pom152 and (2) between Pom152 and Pom34. However, the overall integrity of the complex depends on the presence of both Poms and predominantly relies on interactions that occur on the cytosolic portions of these three transmembrane proteins (Fig. 1 I).
Nup59 and Nup53 directly interact with Ndc1 and also bind directly to Nup157 and Nup170
To discriminate between direct and indirect interactions connecting Ndc1 to its other binding partners, we used in vitro binding approaches with purified proteins. First, we tested whether Ndc1 directly binds to Nup59. In this analysis, we also included the highly related Nup53, which was not detected in the original MS analysis (Fig. 1 A). Tandem affinity purification (TAP)–tagged Ndc1 was purified from yeast cells using S protein–conjugated agarose beads (S beads), whereas recombinant GST fusions of Nup53 and Nup59 were obtained from Escherichia coli (Fig. 2 A). To probe for direct binding, we incubated GST-Nup53 or GST-Nup59 fusion proteins premixed with E. coli extract (1:4 in total protein concentration) with Ndc1–S beads or with S beads alone and eluted bound proteins with high salt. Addition of E. coli extract was used to internally control for nonspecific binding interactions with Ndc1. We found that both GST-Nup53 and GST-Nup59 were specifically purified from the protein mixtures by Ndc1-coated beads (Fig. 2, B and C). These interactions did not depend on the presence of E. coli proteins and were not detected with GST alone (Fig. S1 B), demonstrating that both Nup59 and Nup53 directly and specifically bind to Ndc1.
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-helical domains (AAHDs) at their C termini (Patel and Rexach, 2007), prompting us to test whether the AAHDs are important for Ndc1 binding. We created truncations of both Nup53 and Nup59 and tested them for binding to Ndc1. The removal of the last 15 amino acids completely abolished Nup59 binding to Ndc1 and severely impaired the binding of Nup53. Further deletion of the terminal 50 amino acids completely prevented the interactions of both proteins with Ndc1, demonstrating that the very C termini of Nup53 and Nup59 are necessary for the direct interaction with Ndc1 (Fig. 2, D and E). To examine whether these C-terminal domains are also sufficient for Ndc1 binding, we expressed GST fusion proteins containing the C-terminal 100 amino acids of Nup53 and Nup59. As shown in Fig. 2 (D and E), the short C-terminal fragments of both Nup53 and Nup59 efficiently interacted with Ndc1, which is comparable with full-length proteins. Because Nup53 and Nup59 are homologous proteins that use similar C-terminal domains for Ndc1 binding, it was important to determine whether these two proteins use overlapping or distinct binding sites on Ndc1. To differentiate between these possibilities, Ndc1 beads were premixed either with a 10-fold molar excess of GST or with a 10-fold molar excess of GST-Nup53 and then incubated with GST-Nup59. As shown in Fig. 2 F, preincubation with an excess of GST-Nup53 caused a significant reduction in GST-Nup59 binding when compared with the GST control. Furthermore, the C terminus of Nup53 (Nup53376–475) was sufficient to inhibit the interaction between Nup59 and Ndc1, whereas an N-terminal fragment of Nup53 lacking the AAHD (Nup531–425) had no effect (Fig. 2 G). Conversely, the interaction between Nup53 and Ndc1 was blocked when Ndc1-TAP beads were preincubated with an excess of the C-terminal 100 amino acids of Nup59 (GST-Nup59429–528), but no inhibition was seen in the presence of a Nup59 variant that lacks the extreme C terminus (GST-Nup591–478; Fig. 2 G). These competition experiments show that Nup53 and Nup59 cannot bind to Ndc1 at the same time, suggesting that they use similar or overlapping binding sites. Furthermore, they support a model wherein Ndc1 forms two alternative complexes at the NPC, one with Nup53 and one with Nup59 (Fig. 2 H).
We next tested whether Ndc1 could also directly bind to Nup157 or Nup170, but our attempts to detect specific direct interactions between these proteins were not successful (unpublished data). This suggested that the interactions between Ndc1, Nup157, and Nup170 that we observed in Fig. 1 A might be indirect and mediated by bridging factors. Because Nup53 and Nup59 were previously shown to copurify with Nup170 (Marelli et al., 1998) and Nup170 shares high similarity with Nup157, we explored the pairwise binding interactions of Nup53 and Nup59 with Nup170 and Nup157. TAP-tagged fusions of full-length Nup157 and Nup170 or TAP-tagged alone were purified from yeast (Fig. 3 A) and tested for their ability to bind to maltose-binding protein (MBP) fusions of Nup53 or Nup59. We again included a mixture of E. coli proteins to internally control for binding specificity. Although Nup170 specifically purified both MPB-Nup53 and MBP-Nup59 from the protein mixtures, Nup157 only bound to MBP-Nup53 (Fig. 3 B and Fig. S1 C), suggesting that the interactions between these two pairs of proteins are not identical.
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Nucleoporins interacting with Ndc1 are redundantly required for cell viability
Our biochemical analyses demonstrated that the interaction neighborhood of Ndc1 consists of four nucleoporins, Nup53, Nup59, Pom152, and Pom34. As a first step toward understanding the functional significance of these interactions, we investigated the requirement of these proteins for cell viability. Previous work had shown that single deletions of POM152, POM34, NUP53, or NUP59 and pom152
pom34 or nup53 nup59 double deletions are viable (Marelli et al., 1998; Madrid et al., 2006; Miao et al., 2006). Similarly, combined deletions of NUP53 with either POM34 or with POM152 do not affect viability (Marelli et al., 1998; Miao et al., 2006; Table I). In contrast, cells containing deletions of NUP59 together with either POM152 or POM34 are not viable (Table I; Marelli et al., 1998; Miao et al., 2006). We extended this analysis to all combinations of the four Ndc1-interacting nucleoporins using complete gene knockouts and conditional depletions using repressible GAL or MET promoters (Table I). This analysis confirmed that NUP59 is essential for cell viability in the absence of either POM152 or POM34 independently of whether NUP53 is present or not (Table I and Fig. S2 A). Similarly, using conditional alleles of POM34 or POM152 expressed under control of the GAL promotor, we observed that each of these nucleoporins is essential when both NUP53 and NUP59 are deleted (Table I and Fig. S2 B). These results indicate that two redundant functional elements exist among Ndc1-interacting proteins, Nup59 alone, and Pom152 together with Pom34. In addition, this analysis demonstrates that the essential role of these proteins is independent of Nup53 and that Nup53 is therefore functionally distinct from Nup59.
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pom34 nup53 cells that conditionally express NUP59 (Fig. S3). In the presence of Nup59, Ndc1 correctly localized to NE, but upon Nup59 depletion, it gradually mislocalized into the peripheral ER (Fig. 4 A, i; and Fig. S4 A). Importantly, this effect preceded any significant changes in cell growth, indicating that the mislocalization of Ndc1 is not a secondary consequence of general cell malfunction (compare Fig. 4 A [i] with Fig. S2 C). Similarly, in pom152 pom34 cells, the depletion of Nup59 also resulted in partial mislocalization of Ndc1 (Fig. 3 A, c; and Fig. S2 C). However, in cells in which POM152 and POM34 were not deleted, the depletion of Nup59 did not cause mislocalization of Ndc1 either in the presence or absence of Nup53 (Fig. 4 A, ii and iv), demonstrating that the two Poms can compensate for the loss of Nup59.
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nup59 cells, which express either of the POM genes in a conditional manner and examined the distribution of Ndc1 via GFP. In both cases, Ndc1 predominantly localized to NE in permissive conditions, although some faint peripheral ER localization was already apparent. However, upon shift to nonpermissive conditions, Ndc1 became gradually dispersed from NE (Fig. 4 B, i and iii). This redistribution coincided with the depletion of the respective Poms and started before pronounced defects in cell growth became apparent (compare Fig. 4 B [i and iii] with Fig. S2 D). No mislocalization of Ndc1 was observed in strains in which NUP53 and NUP59 were present (Fig. 4 B, ii and iv). Together, these results present evidence that the essential functions of Nup59 and the Poms tightly correlate with their role in targeting of Ndc1 to the NPC. Consistent with our biochemical findings, this also suggests that Nup59 and an intact Ndc1 subcomplex play a critical but redundant role in establishing the connections between Ndc1 and other core elements of NPC.
Ndc1-interacting nucleoporins play a key role in NPC structure and function
We have previously demonstrated that depletion of Ndc1 in yeast has severe consequences on NPC structure and function (Madrid et al., 2006). To investigate the functional consequences accompanying the mislocalization of Ndc1 that we observed after the removal of its interaction partners, we modified the aforementioned strains to express GFP-tagged versions of various nucleoporins, including the cytoplasmic Nup82, the central Nup188 and Nup133, and the nuclear Nup2. To examine functional defects of NPC, we also monitored the localization of GFP fusions of the nuclear protein Npl3 or its variant Npl3(S411A), which has a weakened nuclear import activity (Siebel and Guthrie, 1996; Gilbert et al., 2001).
We first analyzed the effect of Nup59 depletion in pom152 pom34 strains either in the presence or absence of Nup53. As expected, in pom152 pom34 cells expressing Nup59, all four nucleoporins were properly localized (Fig. 5 A, left). However, upon Nup59 depletion, cells displayed a partial mislocalization of Nup82, Nup133, and Nup2 in foci that appeared to be associated with NE (Fig. 5 A, right). This localization defect correlated with a defect in NPC function because the sensitive nuclear import reporter Npl3(S411A)-GFP partially mislocalized to the cytoplasm (Fig. 5 A).
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pom152 nup53 cells conditionally expressing Nup59, defects in nucleoporin distribution were already evident even in permissive conditions (Fig. 5 B, left). Furthermore, these cells partially mislocalized the strongly imported Npl3 protein (compare Fig. 5 B [left] with Fig. S4 B), indicating that they have both structural and functional NPC defects. Upon depletion of Nup59, these defects were further exacerbated, as indicated by mislocalization of all four nucleoporins and the strong cytoplasmic mislocalization of Npl3-GFP (Fig. 5 B, right). Therefore, in the absence Pom152 and Pom34, both Nup53 and Nup59 become critical for the proper structural organization and function of NPC. However, their roles are not entirely equivalent because only NUP59 is synthetically lethal with POM152 and POM34. In addition, Nup53 and Nup59 have partially redundant roles because we observed exacerbated functional and structural defects in the absence of both these nucleoporins as compared with the conditions when only one of them was absent. We next investigated the effects on nuclear pore organization in nup53 nup59 cells that conditionally express either POM34 or POM152 (Fig. 6). In permissive conditions, we did not observe major localization defects of the reporter nucleoporins in either of these strains (Fig. 6, A and B, left). The only exception was Nup82, which we found to mislocalize in the GAL-3xHA-POM152 cells. Because we did not observe this in the GAL-3xHA-POM34 cells, this phenotype is likely a consequence of the high level of Pom152 that is expressed in the presence of galactose. This had no detectable consequence on NPC function, as the Npl3-GFP reporter efficiently accumulated within the nuclei of these cells (Fig. 6, A and B, left). However, upon depletion of either Pom34 or Pom152, nup53 nup59 cells displayed almost complete mislocalization of Nup82 and a partial mislocalization of the central nucleoporins Nup133 and Nup188 into cytoplasmic foci. At the same time, Nup2 appeared to localize normally. This redistribution of nucleoporins also coincided with a strong mislocalization of Npl3-GFP to the cytoplasm, indicating impaired NPC function (Fig. 6, A and B, right). The similarity in the NPC phenotypes that we observed upon depletion of Pom152 or Pom34 indicates that these proteins cooperate to perform a similar function in the structural organization of NPC. Such cooperativity is consistent with our observations that the integrity of the Ndc1 subcomplex depends on both Pom152 and Pom34.
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pom34 GAL-NUP59 cells grown in permissive conditions displayed no obvious NPC defects (Fig. 7 A, i). However, upon Nup59 depletion, pom152 pom34 cells contained a large fraction of NPCs that had an increase in pore size (Fig. 7, A [ii] and B [i]). A similar pore dilation phenotype could be also observed in pom152 pom34 nup53 MET3-3xHA-NUP59 cells even in the presence of Nup59, which is consistent with our fluorescence microscopy analysis (Fig. 5 B, left). Interestingly, upon depletion of Nup59, the NPC defects became significantly enhanced, and in addition to enlarged pores, we also observed large openings in NE that apparently lack electron-dense material (Fig. 7, A [iv and v] and B [ii]) reminiscent to the phenotype we had previously observed in pom152 cells after the depletion of Ndc1 (Madrid et al., 2006). A similar EM analysis in nup53 nup59 cells that conditionally expressed POM34 did not reveal obvious NPC defects in either permissive or nonpermissive conditions (Fig. 7, A [vi and vii] and B [iii]). However, quantitative comparison of the mean number of pore structures at NE showed that these cells had a significant reduction in pore number and contained only approximately half the number of NPCs per nuclear section (Fig. 7 B, vi).
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Depletion of Pom34 results in a reversible mislocalization of newly synthesized Nup82 in nup53 nup59 cells
The depletion of Pom34 in nup53 nup59 cells resulted in nucleoporin mislocalization (Fig. 6 A), yet our EM analysis did not reveal obvious NPC defects. However, these cells displayed a significant reduction in pore number (Fig. 7). We were therefore interested to investigate whether the mislocalized nucleoporins represent newly synthesized proteins that fail to be incorporated into NPCs or whether they are derived from previously existing NPCs that have disintegrated. To address this question, we developed an assay to monitor the fate of old, already assembled NPCs as well as the assembly of new NPCs. We took advantage of the photoconvertible fluorescent protein Dendra, which irreversibly switches from green to red fluorescence upon the application of a short pulse of UV light (Fig. 8 A; Gurskaya et al., 2006). To test this approach, we fused one of the core nucleoporins, Nup133, with a double Dendra tag (2xDendra) in wild-type cells and followed its distribution before and after photoconversion. Initially, Nup133-2xDendra displayed the characteristic punctate nuclear rim distribution, which was detected exclusively in the green channel without any red signal. However, after the application of a short UV pulse, Nup133-2xDendra was completely photoconverted into the red fluorophore. Over time, newly synthesized Nup133-2xDendra reemerged in the green channel (Fig. 8 B). The appearance of the green signal was dependent on protein synthesis (unpublished data), demonstrating that Nup133 synthesized before photoconversion was exclusively detected in the red channel and thus could be distinguished from newly made protein. We conclude that the Dendra tag enables separate tracking of old and newly synthesized nucleoporins within single living cells and their progeny.
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nup59 cells upon depletion of Pom34 (Fig. 6 A). As expected, in permissive conditions, Nup82-2xDendra properly localized to NPCs (Fig. S4 C). A time course analysis revealed that Nup82 started to mislocalize as early as 6 h of incubation in the nonpermissive conditions (Fig. 8 C), but no prominent mislocalization could be detected at the 4-h time point. We therefore preincubated nup53 nup59 GAL-3xHA-POM34 cells in dextrose-containing medium for 4 h to initiate depletion of Pom34 and then subjected them to photoconversion (Fig. 8 D, left). After an additional 6-h-long chase period, we observed that newly synthesized Nup82 accumulated almost exclusively in cytoplasmic foci. At the same time, previously synthesized Nup82 was still clearly detectable at the NE (Fig. 8 D, right). This shows that in nup53 nup59 cells, Pom34 is required to efficiently incorporate newly synthesized Nup82 into NPCs, suggesting an NPC assembly defect in these cells. To test whether the mislocalization of Nup82 is reversible, NUP82-2xDendra–expressing cells were grown in dextrose-containing medium to induce mislocalization of Nup82. Cells were then placed into galactose-containing medium to reinduce POM34 expression, immediately photoconverted, and reimaged after an additional 3-h chase period (Fig. 8 E). During this chase period, the cytoplasmic foci that contained old Nup82 redistributed into the NE. Furthermore, newly synthesized Nup82 started to correctly localize into nuclear rims (Fig. 8 E). These results suggest that the Pom34-dependent block in NPC formation is reversible and that Nup82, which had accumulated in the cytoplasm, can be chased back into the NE.
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Nup53 and Nup59 have similar patterns of physical interactions in vitro (Fig. 9) and display some functional redundancy because the loss of both proteins leads to an exacerbated perturbation of NPC structure and function in the pom152 pom34 background when compared with the individual loss of each protein (Fig. 5). However, no synthetic lethal interactions between NUP53 and the two POMs can be detected, and NUP53 cannot functionally compensate for the loss of NUP59 (Table I). This suggests that these two highly related proteins have functionally diverged to play distinct roles at the NPC. Consistent with this, we could detect only Nup59 in the Ndc1 purifications from cell extracts, and only Nup59 conferred efficient recruitment of Ndc1 to the NE in pom152 pom34 mutants (Fig. 4). At present, the molecular basis for the functional differences between Nup59 and Nup53 remains unclear.
Role of Ndc1-interacting nucleoporins in NPC biosynthesis and maintenance
The cell growth phenotypes that we observed upon the loss of the various Ndc1-interacting proteins were accompanied by mislocalization of Ndc1 and other structural nucleoporins, ultrastructural NPC abnormalities, and functional NPC defects. In general, the severity of the NPC defects gradually increased upon the loss of multiple proteins, but collectively, the four Ndc1-interacting proteins appear to be important (a) to connect Ndc1 to other structural elements of NPC, (b) to anchor Ndc1 to the pore, and (c) to maintain the structural integrity of NPC. However, the defects within the different mutants varied, and we found that the conditional depletion of Nup59 (in either pom152 pom34 or pom152 pom34 nup53 backgrounds) and the conditional depletion of the Poms (in the nup59 nup53 background) induced distinct phenotypes. For example, in nup59 nup53 cells in which Pom34 was depleted, we observed selective mislocalization of a subset of nucleoporins into cytoplasmic foci. This phenotype was accompanied by a reduction in pore number in the absence of obvious ultrastructural NPC defects (Figs. 6 and 7). To investigate the origin of the mislocalized nucleoporins in these cells, we took advantage of the photoconvertible fluorescent protein Dendra, which enabled us to monitor the intracellular distribution of newly synthesized and preexisting nucleoporins in parallel (Fig. 8). Using Nup82-Dendra, we showed that upon Pom34 repression in nup59 nup53 cells, newly synthesized Nup82 was almost exclusively localized to cytoplasmic foci. This suggests that newly synthesized Nup82 cannot be incorporated into NPCs and that the process of de novo NPC assembly is blocked in these cells. This conclusion is consistent with the observation that these cells have a reduced number of pores (Fig. 7), as these cells can undergo several rounds of division after the shift to the nonpermissive conditions (e.g., Fig. S2 D) and presumably dilute previously synthesized pores between mother and daughter cells. Interestingly, Makio et al. (2009) also describes defects in new NPC biosynthesis in cells simultaneously lacking Nup170 and Nup157. It is therefore likely that the connection to Nup170 and Nup157 established by Ndc1-interacting proteins is critical for new NPC biosynthesis. Notably, the mislocalization of Nup82 was reversible, and the previously synthesized pool of Nup82 that was present in cytoplasmic foci could be chased back to the NE upon reinduction of Pom34 (Fig. 8). An apparent reversible NPC assembly defect was recently also described in apq12 mutants (Scarcelli et al., 2007). However, in this case, the defects in NPC assembly were accompanied by invaginations of the inner nuclear membrane, which we did not observe in our strains.
Surprisingly, when we depleted Nup59 in a pom152 pom34 background, we induced a distinct set of phenotypes. Under these conditions, we did not only observe nucleoporin mislocalization but also found severe ultrastructural defects at NPC. Most strikingly, we detected a significant increase in NPC diameter and, in the absence of Nup53, the appearance of large openings within NE, which is reminiscent to the phenotype that we had previously described upon depletion of Ndc1 in the absence of Pom152. Unfortunately, the Dendra assay did not allow us to unambiguously answer the question of whether these enlarged pores represent blocked NPC assembly intermediates or whether they are derived from destabilized pores that have fallen apart. However, pom152 pom34 GAL-NUP59 cells display an NPC maintenance defect that can be illustrated with the Dendra assay by the mislocalization of already existing nucleoporins in nonpermissive conditions (Fig. S5). Furthermore, all NPC defects that we observe upon Nup59 depletion are irreversible (unpublished data). Therefore, we favor the hypothesis that in these cells, the stability of NPCs is compromised, which ultimately leads to the disintegration of existing pore structures and the formation of large openings within the NE. Future studies will now be required to analyze the molecular details of how Ndc1 and its interacting partners contribute to NPC assembly and maintenance and to dissect the mechanism of how NPCs are inserted into the lipid bilayers of NE.
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Identification of Ndc1-interacting proteins
Pull-downs (Fig. 1 A) were performed using a modification of the protocol described in Carvalho et al. (2006). Cells from 300 ml of culture grown to OD600 = 2.5 were harvested, washed, and subsequently lysed in lysis buffer (LB; 0.1 M Hepes, pH 7.5, 0.3 M KCl, 0.2 mM EDTA, 10 mM EGTA, 20 mM MgOAc, and 10 mM β-mercaptoethanol) in the presence of protease inhibitors using a mini-bead beater (BioSpec Products, Inc.). To prepare crude membrane fractions, the extracts were first subjected to a low-speed centrifugation to remove large cell debris and nonlysed cells followed by high-speed centrifugation (16,000 rpm; SS-34 rotor; Sorvall) for 30 min to pellet membranes. The membranes were extracted in 1.5 ml ice-cold LB/inhibitors plus 1% digitonin (final concentration), and soluble fractions were incubated for 1.5 h at 4°C with 100 µl S protein–conjugated agarose beads (S beads; EMD). Beads were extensively washed, and bound proteins were eluted by 5 min boiling with SDS sample buffer diluted 1:3 with distilled water and separated by SDS-PAGE. Proteins were visualized with GelCode staining reagent (Bio-Rad Laboratories), and the desired areas differing in protein content were excised from the gel and identified by matrix-assisted laser desorption/ionization time of flight MS and LC-MS/MS.
Characterization of the interactions between Ndc1, Pom152, and Pom34
The analytical TAP-tagged affinity pull-downs (Fig. 1, B–D and G) were performed using a procedure similar to the aforementioned one using S beads. Eluted proteins were separated by SDS-PAGE and visualized with SYPRO-Ruby (Invitrogen) using a UV transilluminator coupled to a digital camera (Alpha Innotech). Conditions for TAP-tagged pull-downs at a lower salt concentration (Fig. 1 B) were identical except that experiments were performed in LB diluted 1:1 with distilled water. For Western blotting experiments (Fig. 1 F), digitonin was added to 1 ml bead beater cell lysate up to a final concentration of 1%, and after 10 min incubation on ice, the solubilized material was clarified by 2 min centrifugation at 10,000 rpm in a tabletop mini centrifuge. The extract was incubated for 1 h with 20 µl S beads. The beads were washed and eluted with SDS sample buffer. After resolving by SDS-PAGE, the proteins were blotted onto nitrocellulose membranes and probed with the respective primary and secondary antibodies.
Purification of recombinant proteins and preparation of bacterial cell extract
Cultures of E. coli BL-21 cells transformed with the respective protein-coding plasmids were induced with 0.5 mM IPTG for 2 h at RT. Cells were harvested by centrifugation and lysed by sonication in 1:20 culture volume of LB (PBS, pH 7.4, 1 mM PMSF, and 5 mM DTT) for GST fusions and (50 mM Na-phosphate, pH 7.5, 300 mM NaCl, 1 mM PMSF, and 5 mM β-mercaptoethanol) for 6xHis-MBP fusions. Lysates were supplied with 1% Triton X-100 (final concentration) for GST fusions or 0.5% Triton X-100 for 6xHis-MBP fusions and clarified by 10 min centrifugation at 12,000 rpm (SS-34 rotor). Supernatants were incubated with either Sepharose-GSH beads (GE Healthcare) or His-Select affinity beads (Sigma-Aldrich). Beads were washed with wash buffer (PBS, pH 7.4, 0.1 mM DTT, and 0.1% Tween 20) for GST fusions or (50 mM Na-phosphate, pH 7.5, 300 mM NaCl, 1 mM β-mercaptoethanol, and 0.1% Tween 20) for 6xHis-MBP fusions and eluted with either glutathione elution buffer (75 mM Tris-HCl, pH 8.1, and 10 mM GSH) or with His-tag elution buffer (50 mM Na-phosphate, pH 7.5, 300 mM NaCl, and 200 mM imidazole).
Preparation of Ndc1-TAP, TAP-Nup170, and TAP-Nup157 beads
Yeast cells conferring galactose-inducible overexpression of Ndc1-TAP (KWY2028) were grown in medium containing 2% dextrose, washed, and diluted into medium containing 2% galactose to induce Ndc1-TAP overexpression. Cells were grown overnight and harvested, washed, and lysed in LB containing protease inhibitors in a preparative scale bead beater (BioSpec Products, Inc.). Cell debris was removed from the lysate by centrifugation (5 min at 2,000 rpm in a tabletop centrifuge), and membranes were collected by high-speed centrifugation at 17,000 rpm (SS-34 rotor) for 1 h. The membrane pellet was diluted with ice-cold LB/inhibitors, frozen in liquid N2 in 1-ml aliquots, and stored at –70°C. For preparation of Ndc1-TAP S beads, aliquots of the membrane suspension were diluted 1:1 with LB plus protease inhibitors and 1.2% octyl-D-glucopyranoside (final concentration), incubated for 20 min, and clarified by a 10,000 rpm centrifugation in a tabletop mini centrifuge. To immobilize Ndc1-TAP, the supernatant was then incubated with S beads. Alternatively, Ndc1-TAP membranes were solubilized with 0.5% Triton X-100 and washed with LB plus 0.1% Tween 20. The Triton extraction protocol provided better efficiency of Ndc1 immobilization, although the protein appeared to be less active in the in vitro binding experiments when compared with the octyl-D-glucopyranoside extraction procedure. The overexpression of TAP-Nup157 (KWY2377) and TAP-Nup170 (KWY2378) was similar as described for Ndc1-TAP. Lysis was performed using glass beads with ice-cold LB/inhibitors supplemented with 1% Triton X-100 (final concentration). Insoluble cell debris was removed by 5-min 4,500 rpm centrifugation in a tabletop centrifuge, and the supernatant was incubated with S beads and extensively washed with LB supplemented with 0.1% Tween 20.
In vitro binding assays with Nup53, Nup59, Ndc1, Nup157, and Nup170
For each in vitro binding reaction, aliquots of the bait protein were premixed with an equal volume of LB or LB containing bacterial proteins in the presence of 10% glycerol and 0.4% digitonin, and protein mixtures were added to preequilibrated beads. For competition assays (Fig. 2, F and G), beads were first preincubated with an excess of competitor protein solution followed by the addition of the respective full-length protein. Beads were briefly washed and eluted with high salt elution buffer (1.25 M NaCl, 50 mM Hepes-KOH, pH 7.5, and 0.2% digitonin). Eluted proteins were precipitated with methanol-chloroform, separated by SDS-PAGE, and stained with SYPRO-Ruby. The remaining beads were also subjected to a second stringent elution with ammonium hydroxide (Oeffinger et al., 2007) to exclude the possibility that the binding of some proteins was not detected because they resisted salt elution. The ammonium hydroxide eluates were dried in a Speedvac (GMI, Inc.) and resolubilized in SDS sample buffer before separation by SDS-PAGE.
Fluorescence microscopy
Cells were grown at 30°C in permissive synthetic selection media (containing galactose or containing dextrose and lacking methionine) or in the nonpermissive synthetic selection media (containing dextrose or containing dextrose and 0.2 mg/ml methionine). The media were supplemented with twofold excess of adenine, and cell density was kept under OD600 = 0.5 to reduce autofluorescence. For all live imaging experiments except for color Dendra assays, cells were concentrated by centrifugation, mounted under a cover slide in culture media, and imaged directly. All Images were acquired with a digital camera (CA742-98; Hamamatsu Photonics) controlled by the Metamorph software program (MDS Analytical Technologies). Images were processed using Photoshop (CS2; Adobe), and figures were assembled using Photoshop and Illustrator (CS2; Adobe).
For depletion Dendra experiments, cells were grown overnight in the permissive media essentially as described in the previous paragraph and diluted into the nonpermissive media. Cells were collected for Dendra microscopy at the times specified in Figs. 8 and S5. Cells were washed with fresh medium and concentrated during the final wash step. 2 µl cells was placed on a 2% low melt agarose pad prepared using respective medium. Dendra was converted by 4 x 20–ms pulses of UV light in DAPI channel followed by 8 s YFP exposure to bleach remaining signal. Slides were incubated at 30°C between time points. For recovery experiments, cells were grown in nonpermissive conditions for the time stated in Fig. 8. Cells were washed three times with 500 µl of the permissive media, and 2 µl was placed on an agarose pad prepared using the permissive media. Cells were imaged immediately, incubated at 30°C for the time stated in Fig. 8, and imaged again. Dendra was converted with one 20-ms UV exposure followed by a 10-s YFP exposure to bleach remaining signal. After the chase period, the same field of cells was imaged again in rhodamine and YFP channels.
Ultrastructural analysis
The EM analysis of yeast mutants was performed essentially as described in Madrid et al. (2006).
Online supplemental material
Fig. S1 shows the localization of Ndc1 and Pom34 in strains expressing myc-tagged Pom152 variants and provides additional analyses of the protein interactions of Ndc1. Fig. S2 shows the growth of the various deletion and depletion strains used in this study. Fig. S3 illustrates the functionality of the tagged Ndc1 variants used in this study. Fig. S4 shows the localization of different reporter proteins in various mutants affecting the Ndc1 network. Fig. S5 shows the distribution of old and new Nup133-Dendra protein in pom152 pom34 GAL-3xHA-NUP59 cells upon depletion of Nup59. Tables S1 and S2 describe the plasmids and yeast strains used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200810030/DC1.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health (GM58065 to K. Weis) and by the Carl-Trygger Foundation (T. Kieselbach).
Submitted: 6 October 2008
Accepted: 2 April 2009
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References |
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|---|
Aitchison, J.D., M.P. Rout, M. Marelli, G. Blobel, and R.W. Wozniak. 1995. Two novel related yeast nucleoporins Nup170p and Nup157p: complementation with the vertebrate homologue Nup155p and functional interactions with the yeast nuclear pore-membrane protein Pom152p. J. Cell Biol. 131:1133–1148.
Alber, F., S. Dokudovskaya, L.M. Veenhoff, W. Zhang, J. Kipper, D. Devos, A. Suprapto, O. Karni-Schmidt, R. Williams, B.T. Chait, et al. 2007a. Determining the architectures of macromolecular assemblies. Nature. 450:683–694.[CrossRef][Medline]
Alber, F., S. Dokudovskaya, L.M. Veenhoff, W. Zhang, J. Kipper, D. Devos, A. Suprapto, O. Karni-Schmidt, R. Williams, B.T. Chait, et al. 2007b. The molecular architecture of the nuclear pore complex. Nature. 450:695–701.[CrossRef][Medline]
Antonin, W., C. Franz, U. Haselmann, C. Antony, and I.W. Mattaj. 2005. The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation. Mol. Cell. 17:83–92.[CrossRef][Medline]
Antonin, W., J. Ellenberg, and E. Dultz. 2008. Nuclear pore complex assembly through the cell cycle: regulation and membrane organization. FEBS Lett. 582:2004–2016.[CrossRef][Medline]
Beck, M., F. Forster, M. Ecke, J.M. Plitzko, F. Melchior, G. Gerisch, W. Baumeister, and O. Medalia. 2004. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science. 306:1387–1390.
Beck, M., V. Lucic, F. Forster, W. Baumeister, and O. Medalia. 2007. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature. 449:611–615.[CrossRef][Medline]
Belgareh, N., G. Rabut, S.W. Bai, M. van Overbeek, J. Beaudouin, N. Daigle, O.V. Zatsepina, F. Pasteau, V. Labas, M. Fromont-Racine, et al. 2001. An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. J. Cell Biol. 154:1147–1160.
Berke, I.C., T. Boehmer, G. Blobel, and T.U. Schwartz. 2004. Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex. J. Cell Biol. 167:591–597.
Brohawn, S.G., N.C. Leksa, E.D. Spear, K.R. Rajashankar, and T.U. Schwartz. 2008. Structural evidence for common ancestry of the nuclear pore complex and vesicle coats. Science. 322:1369–1373.
Carvalho, P., V. Goder, and T.A. Rapoport. 2006. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell. 126:361–373.[CrossRef][Medline]
Chial, H.J., M.P. Rout, T.H. Giddings, and M. Winey. 1998. Saccharomyces cerevisiae Ndc1p is a shared component of nuclear pore complexes and spindle pole bodies. J. Cell Biol. 143:1789–1800.
Cronshaw, J.M., A.N. Krutchinsky, W. Zhang, B.T. Chait, and M.J. Matunis. 2002. Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158:915–927.
D'Angelo, M.A., and M.W. Hetzer. 2006. The role of the nuclear envelope in cellular organization. Cell. Mol. Life Sci. 63:316–332.[CrossRef][Medline]
Dultz, E., E. Zanin, C. Wurzenberger, M. Braun, G. Rabut, L. Sironi, and J. Ellenberg. 2008. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J. Cell Biol. 180:857–865.
Franz, C., R. Walczak, S. Yavuz, R. Santarella, M. Gentzel, P. Askjaer, V. Galy, M. Hetzer, I.W. Mattaj, and W. Antonin. 2007. MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly. EMBO Rep. 8:165–172.[CrossRef][Medline]
Gilbert, W., C.W. Siebel, and C. Guthrie. 2001. Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation. RNA. 7:302–313.[Abstract]
Gillespie, P.J., G.A. Khoudoli, G. Stewart, J.R. Swedlow, and J.J. Blow. 2007. ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing. Curr. Biol. 17:1657–1662.[CrossRef][Medline]
Gurskaya, N.G., V.V. Verkhusha, A.S. Shcheglov, D.B. Staroverov, T.V. Chepurnykh, A.F. Fradkov, S. Lukyanov, and K.A. Lukyanov. 2006. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24:461–465.[CrossRef][Medline]
Hallberg, E., R.W. Wozniak, and G. Blobel. 1993. An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region. J. Cell Biol. 122:513–521.
Harel, A., A.V. Orjalo, T. Vincent, A. Lachish-Zalait, S. Vasu, S. Shah, E. Zimmerman, M. Elbaum, and D.J. Forbes. 2003. Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores. Mol. Cell. 11:853–864.[CrossRef][Medline]
Hawryluk-Gara, L.A., M. Platani, R. Santarella, R.W. Wozniak, and I.W. Mattaj. 2008. Nup53 is required for nuclear envelope and nuclear pore complex assembly. Mol. Biol. Cell. 19:1753–1762.
Hetzer, M.W., T.C. Walther, and I.W. Mattaj. 2005. Pushing the envelope: structure, function, and dynamics of the nuclear periphery. Annu. Rev. Cell Dev. Biol. 21:347–380.[CrossRef][Medline]
Hinshaw, J.E., B.O. Carragher, and R.A. Milligan. 1992. Architecture and design of the nuclear pore complex. Cell. 69:1133–1141.[CrossRef][Medline]
Hodel, A.E., M.R. Hodel, E.R. Griffis, K.A. Hennig, G.A. Ratner, S. Xu, and M.A. Powers. 2002. The three-dimensional structure of the autoproteolytic, nuclear pore-targeting domain of the human nucleoporin Nup98. Mol. Cell. 10:347–358.[CrossRef][Medline]
Hsia, K.C., P. Stavropoulos, G. Blobel, and A. Hoelz. 2007. Architecture of a coat for the nuclear pore membrane. Cell. 131:1313–1326.[CrossRef][Medline]
Jaspersen, S.L., T.H. Giddings, Jr., and M. Winey. 2002. Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p. J. Cell Biol. 159:945–956.
Jeudy, S., and T.U. Schwartz. 2007. Crystal structure of nucleoporin Nic96 reveals a novel, intricate helical domain architecture. J. Biol. Chem. 282:34904–34912.
Longtine, M.S., A. McKenzie, III, D.J. Demarini, N.G. Shah, A. Wach, A. Brachat, P. Philippsen, and J.R. Pringle. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14:953–961.[CrossRef][Medline]
Madrid, A.S., J. Mancuso, W.Z. Cande, and K. Weis. 2006. The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function. J. Cell Biol. 173:361–371.
Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly. J. Cell Biol. 185:459–473.
Mansfeld, J., S. Guttinger, L.A. Hawryluk-Gara, N. Pante, M. Mall, V. Galy, U. Haselmann, P. Muhlhausser, R.W. Wozniak, I.W. Mattaj, et al. 2006. The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells. Mol. Cell. 22:93–103.[CrossRef][Medline]
Marelli, M., J.D. Aitchison, and R.W. Wozniak. 1998. Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p. J. Cell Biol. 143:1813–1830.
Marelli, M., C.P. Lusk, H. Chan, J.D. Aitchison, and R.W. Wozniak. 2001. A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope. J. Cell Biol. 153:709–724.
Maul, G.G., H.M. Maul, J.E. Scogna, M.W. Lieberman, G.S. Stein, B.Y. Hsu, and T.W. Borun. 1972. Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle. J. Cell Biol. 55:433–447.
McDonald, K., and T. Muller-Reichert. 2002. Cryomethods for thin section electron microscopy. Methods Enzymol. 351:96–123.[Medline]
Melcak, I., A. Hoelz, and G. Blobel. 2007. Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding. Science. 315:1729–1732.
Miao, M., K.J. Ryan, and S.R. Wente. 2006. The integral membrane protein Pom34p functionally links nucleoporin subcomplexes. Genetics. 172:1441–1457.
Oeffinger, M., K.E. Wei, R. Rogers, J.A. Degrasse, B.T. Chait, J.D. Aitchison, and M.P. Rout. 2007. Comprehensive analysis of diverse ribonucleoprotein complexes. Nat. Methods. 4:951–956.[CrossRef][Medline]
Osmani, A.H., J. Davies, H.L. Liu, A. Nile, and S.A. Osmani. 2006. Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans. Mol. Biol. Cell. 17:4946–4961.
Patel, S.S., and M.F. Rexach. 2007. Discovering novel interactions at the nuclear pore complex using Bead Halo: A rapid method for detecting molecular interactions of high and low affinity at equilibrium. Mol. Cell. Proteomics. 7:121–131.[CrossRef][Medline]
Rabut, G., P. Lenart, and J. Ellenberg. 2004. Dynamics of nuclear pore complex organization through the cell cycle. Curr. Opin. Cell Biol. 16:314–321.[CrossRef][Medline]
Rasala, B.A., A.V. Orjalo, Z. Shen, S. Briggs, and D.J. Forbes. 2006. ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division. Proc. Natl. Acad. Sci. USA. 103:17801–17806.
Rout, M.P., J.D. Aitchison, A. Suprapto, K. Hjertaas, Y. Zhao, and B.T. Chait. 2000. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148:635–651.
Scarcelli, J.J., C.A. Hodge, and C.N. Cole. 2007. The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes. J. Cell Biol. 178:799–812.
Schrader, N., P. Stelter, D. Flemming, R. Kunze, E. Hurt, and I.R. Vetter. 2008. Structural basis of the nic96 subcomplex organization in the nuclear pore channel. Mol. Cell. 29:46–55.[CrossRef][Medline]
Siebel, C.W., and C. Guthrie. 1996. The essential yeast RNA binding protein Np13p is methylated. Proc. Natl. Acad. Sci. USA. 93:13641–13646.
Stavru, F., B.B. Hulsmann, A. Spang, E. Hartmann, V.C. Cordes, and D. Gorlich. 2006a. NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes. J. Cell Biol. 173:509–519.
Stavru, F., G. Nautrup-Pedersen, V.C. Cordes, and D. Gorlich. 2006b. Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells. J. Cell Biol. 173:477–483.
Suntharalingam, M., and S.R. Wente. 2003. Peering through the pore. Nuclear pore complex structure, assembly, and function. Dev. Cell. 4:775–789.[CrossRef][Medline]
Tcheperegine, S.E., M. Marelli, and R.W. Wozniak. 1999. Topology and functional domains of the yeast pore membrane protein Pom152p. J. Biol. Chem. 274:5252–5258.
Tran, E.J., and S.R. Wente. 2006. Dynamic nuclear pore complexes: life on the edge. Cell. 125:1041–1053.[CrossRef][Medline]
Unwin, P.N., and R.A. Milligan. 1982. A large particle associated with the perimeter of the nuclear pore complex. J. Cell Biol. 93:63–75.
Walther, T.C., A. Alves, H. Pickersgill, I. Loiodice, M. Hetzer, V. Galy, B.B. Hulsmann, T. Kocher, M. Wilm, T. Allen, et al. 2003. The conserved Nup107-160 complex is critical for nuclear pore complex assembly. Cell. 113:195–206.[CrossRef][Medline]
Weirich, C.S., J.P. Erzberger, J.M. Berger, and K. Weis. 2004. The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore. Mol. Cell. 16:749–760.[CrossRef][Medline]
Winey, M., D. Yarar, G.T.H. Jr, and D.N. Mastronarde. 1997. Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes. Mol. Biol. Cell. 8:2119–2132.
Wozniak, R.W., and G. Blobel. 1992. The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope. J. Cell Biol. 119:1441–1449.
Wozniak, R.W., G. Blobel, and M.P. Rout. 1994. POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope. J. Cell Biol. 125:31–42.
Yang, Q., M.P. Rout, and C.W. Akey. 1998. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Mol. Cell. 1:223–234.[CrossRef][Medline]
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