Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in ciliate Tetrahymena

The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of about 30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type, or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally-active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins, and subcellular localization analysis of GFP fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on compositional and structural specificity of NPCs. Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le INTRODUCTION Ciliated protozoa maintain two distinct nuclei within the same cytoplasm: a somatic macronucleus (MAC) and a germline micronucleus (MIC) (Fig. 1A) (Eisen et al., 2006; Orias et al., 2011; Karrer, 2012). The polyploid MAC is transcriptionally active, and its acentromeric chromosomes segregate during cell division by a spindle-independent amitotic process. In contrast, the diploid MIC has transcriptionally inert, centromeric chromosomes that segregate by canonical mitosis. In Tetrahymena thermophila, DNA replication in the MIC and MAC occurs during non-overlapping periods in the cell cycle. Thus, nuclear dimorphism in ciliates involves non-equivalent regulation of multiple activities in two distinct nuclei (Orias, 2000; Goldfarb and Gorovsky, 2009). This is likely to require targeted transport of components to the MIC vs. MAC, for which differences in the NPCs may be important determinants. Previously, we analyzed 13 Tetrahymena nucleoporins (Nups), and discovered that four paralogs of Nup98 were differentially localized to the MAC and MIC (Iwamoto et al., 2009). The MACand MIC-specific Nup98s are characterized by Gly-Leu-Phe-Gly (GLFG) and Asn-Ile-Phe-Ans (NIFN) repeats, respectively, and this difference is important for the nucleus-specific import of linker histones (Iwamoto et al., 2009). The full extent of compositional differentiation of MAC and MIC NPCs could not, however, be assessed, since only a small subset of the expected NPC components were detected. NPCs have been studied in eukaryotes including rat (Cronshaw et al., 2002), Saccharomyces cerevisiae (Rout et al., 2000), Aspergillus nidulans (Osmani et al., 2006), Schizosaccharomyces pombe (Asakawa et al., 2014), Arabidopsis thaliana (Tamura et al., 2010), and Trypanosoma brucei (Degrasse et al., 2009; Obado et al., 2016) (Table S1). The NPC structure has an 8-fold rotational symmetry, and is made up of roughly 30 known Nups organized into subcomplexes (Alber et al., 2007; Bui et al., 2013) (Fig. S1). The Nup93 complex in mammalian cells (Nic96 in S. cerevisiae) forms a stable scaffold composed of Nup93 ScNic96 , Nup205 ScNup192 , Nup188 ScNup188 , Nup155 ScNup170 or ScNup157 , and Nup53/Nup35/MP-44 ScNup53 or ScNup59 (Grandi et al., 1997; Hawryluk-Gara et al., 2005; Amlacher et al., 2011). A second stable scaffold in mammals, the Nup107-160 complex (called the Y-complex or Nup84 complex in S. cerevisiae) is composed of conserved subunits Nup107 ScNup84 , Nup160 ScNup120 , Nup133 ScNup133 , Nup96 ScNup145C , Nup85 ScNup85 , Seh1, and Sec13, together with species-specific subunits (Siniossoglou et al., 1996; Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le Lutzmann et al., 2002; Loiodice et al., 2004). Peripheral to the scaffolds are Phe-Gly (FG) repeat-bearing Nups, whose disordered FG-repeat regions constitute the central channel, with FG repeats interacting with nuclear transport receptors (Terry and Wente, 2009). Three transmembrane (TM) Nups anchoring the NPC to the mammalian nuclear membrane are NDC1, gp210, and POM121 (Greber et al., 1990; Hallberg et al., 1993; Stavru et al., 2006) (in yeast: Ndc1, Pom152, and Pom34 (Winey et al., 1993; Wozniak et al., 1994; Miao et al., 2006)). A distinct nucleoplasmic basket is formed with Tpr ScMlp1/Mlp2 (Cordes et al., 1997; Strambio-de-Castillia et al., 1999). Based on prior analysis, T. thermophila appeared to lack homologs of many widely conserved NPC components. These included scaffold Nups (mammalian Nups205, 188, 160, 133, 107, 85, and 53, among others) from the Nup93 and Y-complexes. Similarly, homologs of FG-Nups Nup214, 153, 62, and 58 were also not detected, as were TM Nups except for gp210. These NPC components may have evaded homology-based searches due to extensive sequence divergence, given the large evolutionary distance between ciliates and animals, fungi, and plants. To address these ambiguities and to better understand NPC differentiation in T. thermophila, we attempted comprehensive identification of Nups. First, we analyzed proteins affinity-captured with known Nups. Furthermore, we mined updated genome and protein databases for characteristic Nup sequences or conserved domains, using in silico structure prediction. The resulting expanded catalog of Tetrahymena Nups, combined with localization data, sheds new light on the extent to which NPC architecture can vary within a single species, and even in a single cytoplasm. RESULTS The Nup93 complex includes a unique Nup205 ortholog and a novel central channel FG-Nup In mammalian cells, the Nup93 complex (Fig. 1B) is composed of Nup93, Nup205, Nup188, Nup155, and Nup53 (Fig. S1) (Grandi et al., 1997; Hawryluk-Gara et al., 2005). In Tetrahymena, we previously identified homologs for Nup93 (TtNup93; Gene Model identifier TTHERM_00622800) and Nup155 (TtNup155; TTHERM_00760460), and found them distributed to MAC and MIC NPCs (Iwamoto et al., 2009). To identify other Nup93 complex components, we used mass spectrometry to analyze anti-GFP Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le immunoprecipitates from Tetrahymena expressing GFP-TtNup93 (Fig. 1C). All of the proteins listed in Table S2 as ‘hypothetical protein’ were examined by Blast search for similarities to known Nups of other species. In addition, all of the ‘hypothetical proteins’ were examined by expression profile analysis in the Tetrahymena Functional Genomics Database (TetraFGD) web site (http://tfgd.ihb.ac.cn/) (for details see the “Microarray” page of the TetraFGD: http://tfgd.ihb.ac.cn/tool/exp (Miao et al., 2009)) (also see Materials and Methods). When either the Blast search or the expression profile analysis (details described below) found similarities to any known Nups, we examined its subcellular localization in T. thermophila by ectopically expressing GFP fused proteins. By these analyses we found Nup308 (TTHERM_00091620) and the novel protein TTHERM_00194800 (TtNup58: Nup58 in Fig. 1D and Table S2). Nup308, a protein of 2675 amino acid residues, was previously identified as a Tetrahymena-specific Nup, but it was not assigned to a subcomplex (Iwamoto et al., 2009). Based on PSIPRED analysis, Nup308 is composed of GLFG repeats forming an N-terminal disordered structure (residues 1–570), followed by a large C-terminal α-helix-rich region (residues 571–2675) (Fig. 2). To identify potential Nup308 counterparts, we looked for Nups in other species with similar distributions of secondary structures. Interestingly, a large α-solenoid domain is a predicted feature of both Nup205 and Nup188, conserved core members of the Nup93 complex (Kosova et al., 1999; Andersen et al., 2013), although these proteins do not have FG repeats. To investigate whether this structural similarity between Tetrahymena Nup308 and Nup205 and Nup188 homologs in other species reflected shared evolutionary origins, we performed a phylogenetic analysis. Nup308 formed a clade with Nup205 orthologs, supported by a bootstrap probability of 72%, but not with Nup188 orthologs (Fig. S2). Nup188 appears absent in Tetrahymena, since we failed to find any candidates in either the database or in our mass spectrometry data. Taken together, our results strongly suggest that Nup308 belongs to the Nup93 complex and is orthologous to human Nup205, but has acquired an unusual GLFG repeat domain. Consistent with this assignment, GFP– Nup308 localized similarly to GFP–TtNup93, being equally distributed between MAC and MIC NPCs (Iwamoto et al., 2009). The second Nup candidate identified in TtNup93 pulldowns was TTHERM_00194800. This small protein (45 kDa deduced molecular weight) is Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le composed of an N-terminal FG-repeat region and a C-terminal coiled-coil region (Fig. 2), which are characteristics of central channel FG-Nups that are tethered by Nup93 (Chug et al., 2015). The secondary structure characteristics of the novel Tetrahymena Nup are highly similar to those of Nup62 and Nup58, central channel proteins in yeast and vertebrates that interact with Nup93 (Grandi et al., 1993, 1997). Because another protein was found as an Nup62 ortholog (described below), this protein is the likely Tetrahymena ortholog of Nup58; therefore, we named it TtNup58 (Nup58 in Fig. 1D,E). Newly identified members of the Y-complex are likely homologs of conserved Nups The vertebrate’s Y-complex (Fig. 3A) contains 10 distinct proteins (Orjalo et al., 2006; Mishra et al., 2010), of which 3 had identified homologs in Tetrahymena (TtSeh1, TtSec13, TtNup96) (Iwamoto et al, 2009). To investigate whether the remaining seven are present in Tetrahymena but had been overlooked due to sequence divergence, we carried out mass spectrometric analysis of anti-GFP immunoprecipitates from cells expressing the known Y-complex GFP-tagged Nups described below. First, in precipitates of GFP–TtSeh1, we identified an 86 kDa protein orthologous to Nup85 (Table S3) with a short stretch of four predicted -strand blades at the N-terminus followed by an -solenoid domain (Fig. 2). That architecture is typical of Nup85 orthologs that are Y-complex components in other organisms (Brohawn et al., 2008). We therefore tentatively named the Tetrahymena protein TtNup85. GFP–TtNup85 localized to NPCs in both the MAC and MIC (Figs 3B and S3A). We then immunoprecipitated GFP–TtNup85, and identified two novel candidate Y-complex core members. Both proteins are composed of a -strand-rich N-terminal half and an -helical-rich C-terminal half. This domain architecture is characteristic of the Y-complex components Nup160 and Nup133 (Berke et al., 2004; Devos et al., 2004), and we tentatively named the Tetrahymena proteins TtNup160 and TtNup133 (Fig. 2 and Table S4). GFP–TtNup160 and GFP–TtNup133 localized to NPCs in both nuclei, like other Y-complex components (Figs 3B and S3A). Another conserved Y-complex component is Nup107, which interacts with Nup96. To search for the Tetrahymena homolog we used GFP–TtNup96 as bait and identified a Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le 109 kDa protein (Table S5) that is rich in predicted -helices like human Nup107 (Fig. 2). The protein, tentatively named TtNup107, localized as a GFP-tagged construct to NPCs of both nuclei (Figs 3B and S3A). The genes encoding all members of the Y-complex except for Nup96 are co-expressed and exhibit sharp expression peaks at 2 h (C-2) after two cell strains with different mating-types were mixed for conjugation (for details see the “Microarray” page of the TetraFGD: http://tfgd.ihb.ac.cn/tool/exp (Miao et al., 2009)) (Fig. 3C). In contrast, TtNup96 exhibits an expression peak at 4 h (C-4). This difference in the timing of expression between TtNup96 and the other Y-complex components may be related to a unique aspect of TtNup96 gene structure: TtNup96 is expressed as part of a single transcription unit together with MicNup98B, under the promoter of the MicNup98B gene (Iwamoto et al., 2009). Three other components of the human Y-complex were not detected in our studies: Nup43, Nup37, and ELYS. These components may be species-specific (Bilokapic and Schwartz, 2012; Rothballer and Kutay, 2012), and genuinely absent from Tetrahymena. They are also absent from S. cerevisiae (Alber et al., 2007) (see Table S1), supporting this idea. Y-complex components show biased localization to the MIC As previously reported, GFP-tagged Nup93 complex members and some of the central channel Nups (TtNup93, TtNup308, and TtNup54) were distributed equally between MAC and MIC NPCs, judging by fluorescence intensities (Iwamoto et al., 2009). In striking contrast, all Y-complex components so far identified exhibit distinctively biased localization to the MIC nuclear envelope (NE) compared to the MAC NE (Fig. 3B). Fluorescence intensities in the MIC were 2.69–3.96 times higher than those in the MAC (Fig. 3B). This biased localization of Y-complex components may be caused by overexpression of the components due to ectopically expressing the GFP-tagged proteins in addition to the expression of endogenously untagged ones. To address this issue, we examined the localization of Nup160-GFP, Nup133-GFP, and Seh1-mCherry expressed from endogenous loci under the control of their native promoters, and therefore expressed at physiological levels. All three proteins showed biased localization, as found for the overexpressed GFP-tagged proteins (compare the images in Fig. 3B and Fig. S3B), Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le suggesting that the biased localization is not caused by overexpression of the tagged proteins. Because the NPC density is similar in the MAC and MIC (Fig. S1 in Iwamoto et al. (2009)), the relative concentration of Y-complex components in the MIC NE suggests that the Y-complex is present at higher copy number per NPC in the MIC compared to the