Copyright Landes. Comparative Genomics, Evolution and Origins of the Nuclear Envelope and Nuclear Pore Complex.

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1 [Cell Cycle 3:12, ; December 20]; 20 Les Bioscience Report Comparative Genomics, Evolution Origins of the Nuclear Envelope Nuclear Pore Complex 20 Les mbiobioscience ce Ben J. Mans Vivek Anantharaman L. Aravind* Eugene V. Koonin* National Center for Biotechnology Information; National Institutes of Health; Bethesda, Maryl USA These authors contributed equally to this work. *Correspondence to: L. Aravind or Eugene Koonin; National Center for Biotechnology Information; National Institutes of Health; Bethesda, Maryl USA; Received 10/18/; Accepted 10/20/ Previously published online as a Cell Cycle E-publication: KEY WORDS nuclear pore, nuclear envelope, karyopherins, nucleoporins, eukaryotic, evolution, pre k a ryo t e, horizontal gene transfer, evolutionary parsimony ACKNOWLEDGEMENTS E.V.K. is grateful to Valerie Doye, Eric Bapteste, Celine Bro c h i e r, John Fuerst, Gaspar Je k e l y, Purificacion Lopez-Garcia, other participants of the meeting on the Origin of Eu k a ryo t i c Nucleus, organized by Patrick Fo rt e r re at La Fondation des Treilles (Tourtour, France) in July 20, for inspiring discussions which were indispensable for the completion of this manuscript. NOTE nd*p rk.g h.gov2 Supplementary material accompanies this manuscript. Figure S1, Figure S2, Table S1 Table S2 can be found at: manscc3-12-sup.pdf ABSTRACT The presence of a distinct nucleus, the compartment for confining the genome, transcription RNA maturation, is a central ( eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. We investigated the provenance of the conserved domains found in these perinuclear proteins reconstructed a parsimonious scenario for NE NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1,, probably, also the key domains of karyopherins nucleoporins, the HEAT/ARM WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late Lelated horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mrna-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. It is shown that several NPC proteins containing super-structureforming α-helical β-propeller modules are most closely related to corresponding proteins in the cytoplasmic vesicle biogenesis coating complexes. From these observations, we infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the prekaryote ) ryo as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding BA Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred ed to have been integral perinuclear components in the las common ancestor of modern eukaryotes. INTRODUCTION The nucleus, the defining feature of the eukaryotic superkingdom, segregates the genome from the cytoplasm. Within the nucleus, the eukaryotic genome is packaged into one or more linear chromosomes, the nucleus is the site of DNA replication, RNA transcription processing (including mrna splicing), ribosome maturation. 1,2 This allows eukaryotes to decouple transcription from translation, which appears pears to be a necessity if translation of unprocessed, intron-containing premrnas is to be ceided, avoided, to regulate gene expression at both transcriptional post-transcriptional level, prov ev i ding for an added level of complexity that is not apparent in prokaryotes. The requirement for physical integrity of the nucleus is, essentially, fulfilled by the nuclear membrane the matrix or lamina. 3,4 Inasmuch as mrnas transcribed processed in the nucleus are translated in the cytoplasm, the nucleus needs a sophisticated means of communication ensuring both import of proteins from the cytoplasm into the nucleus export of mrnas, trnas, ribosomes from the nucleus to the cytoplasm. This role is fulfilled 1612 Cell Cycle 20; Vol. 3 Issue 12

2 by the nuclear pore complex (NPC) which functions as both import er exporter. 5-7 The nuclear lamina tightly associates with the NPC binds to several nuclear envelope (NE) proteins. Thus, it is appropriate to consider the NE, the lamina, the NPC as components of a single, integrated system. The NPC is a distinct, large structure ( MDa) which consists of proteins interacting in various ways CDa) Cto form a complex with eight-fold symmetry in the plane of the NE. Integral ICIntegral pore membrane proteins (Poms) anchor the NPC inside the NE form a channel which surrounds an 8-spoke ring complex consisting of abundant nucleoporin proteins (Nups). The central core of the NPC shows two-fold symmetry with respect to the axes perpendicular to the NE is composed of two halves positioned back-to-back in the midplane of the NE. At the molecular level, the symmetry of the NPC is manifest in the presence of multiple copies of the Nups with 1,2 or 4 copies per spoke, accordingly, 8, 16 or 32 copies per NPC. The NPC has a distinct internal architecture, with specific localization of different ent sub-complexes. 5,8-11 Eight cytoplasmic filaments extend from the NPC into the cytoplasm whereas, on the nucleoplasmic side, the eight filaments are joined by a ring structure to form the so-called nuclear basket. 12 The filaments s e rve as docking sites for transport proteins, ptp in2ins, 2ear a gradient of binding affinities of the transporters (karyopherins) to various Nups defines the transport direction. The karyopherins, also known as importins exportins, serve as transporters of cargo through the NPC. Karyopherins bind to the expoles cargo molecules via nuclear localization signals (NLS) for import into the nucleus to nuclear export signals (NES) for export out of the nucleus. 13,14 The original karyopherins identified were Lout Kapαα Kapβ which form a transport complex, with Kapα recognizing 20 p rotein binds to mrna but needs complex formation with NXT1/p15 (Vertebrates) or Mtr2 (S. cerevisiae) for docking to the N P C. 3 0 Binding of mrna to Mex67 is mediated by a host of proteins involved in mrna processing transcription. The THO/Trex complex (Hpr1, Tho2, Mft1, Thp2) involved in transcription forms a complex with Sub2 Yra1 (UAP56 Aly in animals, respectively), this whole complex is required for effective mrna transport by Mex Sus1, a functional component of the SAGA histone acetylase complex, associates with the Sac3-Thp1 complex which is also involved in mrna export via Mex67. 33,36 Furthermore, the export of the ribosomal subunits, which assemble in the nucleolus, requires participation of several large macromolecular complexes whose organization exact roles are only starting to be elucidated. 37,38 Clearly, nuclear export is intimately linked to transcription, RNA processing, ribosome assembly, these interactions should be taken into account when evolution of the NPC is investigated. Consistently with the coupling of transcription, RNA processing, export, the chromosomes are peripherally tethered to the NE,, in particular, transcriptionally active zones of chromatin tend to associate with the nuclear periphery The outer layer of the NE is contiguous with the ER, while the inner layer is linked via specific integral membrane proteins to the nuclear lamina. 43 The NE lumen also appears to contain certain critical soluble proteins, such as the AAA+ ATPase torsin, which might act as a chaperone regulating the assembly of NE-associated complexe s. 4 4, 4 5 Se veral integral membrane proteins of the inner NE have been characterized in animals; these proteins are, largely, involved in tethering the NE to components of Lp the lamina chromatin, in particular, the lamins BAF (Barrier to Autointegration factor) a classic NLS comprised of basic amino acids, Kapβ docking at Recent proteomic analysis of the mammalian NPC produced an the NPC Nu m e rous additional karyopherins of the Kapβ f a mily extensive ata catalog of NPC subunits substantially improved the have been discovered shown to dock at the NPC recognize existing knowledge of the NPC composition 49 (Cronshaw et al., other NLS versions (Chook YM, Blobel G, 2001). Kapβ proteins are 2002). 02). Likewise, a combination of proteomics, 50 genetic biochemical analysis of the the nematode Unc-84 system, 51,52 large proteins ( kda) containing multiple, tem α-helical HEAT/ARM repeats In yeast, 14 members of the K a pβ f a m i l y genetic analysis of human diseases caused by mutations in genes h a ve been described of which 13 have known functions. In mammals, encoding NE proteins 53,54 have furnished detailed catalogs of NE over 20 members have been described, with no function so far components. eswe e took advantage of these other molecular studies assigned to several of them. 14 The Kapα family proteins have a re l a ted to perform exhaustive phylogenomic analysis of the NE NPC but distinct superhelical structure which consists of eight HEAT/ components. Evolution Bphylogeno on of the NE NPC is a quintessential ARM repeats of ~40 amino acid residues each. 19 Darwinian problem whereby eby emergence of a complex biological Transport of cargo by the karyopherins is regulated by the Ran- device which, at least superficially, appears to be functional only in GTP system. 22,23 During import from the cytoplasm, karyopherins its complete form, has to be explained through a sequence of small, their cargo dock to the NPC translocate across the pore. plausible steps, each of which increases the fitness of the organism. 55 Once in the nucleus, Ran-GTP binds to this complex dissociates He re, we break this problem into two main parts, namely, the distribution of NE NPC components the cargo from the karyopherin. During export, the cargo-karyopherin complex binds to Ran-GTP translocates across the NPC, the presence of homologs in prokaryotes. otes. The former part gives sccross across the eukaryotic kingdom after which Ran-GTP is activated by Ran GTPase activating protein an indication of the probable composition of the minimal NPC (RanGAP), assisted by the Ran-binding protein (RanBP1), to NE, which might have been present in the Last Eu k a ryo t i c hydrolyze the bound GTP, which leads to release of the cargo. Thus, Common Ancestor (LECA), lineage-specific gene innovation, a gradient of Ran-GTP is required for effective transport, with high loss, expansion which occurred in the course of subsequent concentrations of RanGTP in the nucleus low concentrations in evolution. The latter part of the study helps delineating cecontri- of the butions of different prokaryotic lineages to the origin contri- the cytoplasm. Such gradient is maintained through import of cenpc of R a n-gdp into the nucleus in association with nuclear transport NE. From the data gathered through this two-pronged analysis, eis, we f a ctor 2 (NTF2), after which it undergoes exchange of GDP for attempt to infer parsimonious scenarios for origin evolution of GTP, mediated by guanine exchange factor (RanGEF) the components of the NE, NPC, the nuclear compartment Apart from the importin β-ran-dependent transport pathways, itself. other, Ran-independent pathways of nucleocytoplasmic transport have been described. The Mex67/TAP transport pathway is involved in the export of mrna from the nucleus. 27,28,29 The Mex67/TAP La nde des ustibioscience ios scie ien ce s a Cell Cycle 1613

3 Figure 1. Domain architectures of the protein components of the nuclear pore complex the nuclear envelope. The domains are grouped by their occurrence in distinct functional subsystems associated with the NE the NPC. TMR, transmembrane region. Cop opy pyri yrig righ ght Lan nde des Bioscience ien ce MATERIALS AND METHODS The sequences of the characterize d p rotein components of the NE/NPC from human yeast associated proteins were extracted from GenBank. Homologs of each of these proteins were detected t h rough search of the nonredundant protein sequence database (National Center for Biotechnology Information, NIH, Be t h e s d a ) using the BLASTP pro g r a m. 241 The analysis was performed with the annotated protein sequences from 28 completely sequenced e u k a ryotic genomes. Genomes analyze d included the nucleomorph of Gu i l l a rd i a t h e t a (Gt), from Kinetoplastida, L e i s h m a n i a m a j o r (Lm), from the Di p l o m o n a d i d a, Gi a rdia lamblia ( Gl), the Alve o l a t a, Cryptosporidium parvum (Cp), Plasmodium falciparum (Pf) Plasmodium yoelii (Py). From plants, the unicellular red alga Cy a n i d i o s c h y zon mero l a e (Cm) the flowering plants Arabidopsis thaliana (At) Oryza sativa ( Os). The amoeba, Entamoeba histolytica (Eh) the slime mold, D i c t yostelium discoideum ( D d ). From the Fungi, Schizosaccharomyces pombe (Sp), Neurospora crassa (Nc), Magnaporthe g r i s e a (Mg), Aspergillus nidulans ( A n ), Gi b b e rella ze a e (Gz), Sa c c h a ro m yces cere v i s iae (Sc), Cida albicans (Ca) the m i c rosporidia, En c e p h a l i t o zoon cuniculi ( Ec ). From nematodes, Caenorhabditis elegans (Ce) from arthropods, the mosquitoe, Anopheles gambiae (Ag) the fly, Drosophila melanogaster (Dm). The uro- chordate, Ciona intestinales (Ci). The verte- ostakifugu brates, Takifugu rubripes (Fr), Danio rerio (Dr), sco Mus musculus (Mm), Rattus norve g i c u s (Rn) Homo sapiens (Hs). Orthologous relationships ips between known pre d i c ted NPC components encoded in these genomes we re validated using the re c i p rocal best hit approach. enin 242 cases when an ortholog of an NPC component could not be identified in a particular genome, additional searches were performed, using the PSI-BLAST program in order to identify potential orthologs with cesequenc low sequence c o nservation, using the TBLASTN eas program to search untranslated genomic sequences, if available, EST sequences, to account for the possibility that the c o rresponding orthologous gene remains unide n t i f i e d. 241, 243 Transmembrane helices were identified using the TMHMM2 244 TMPRED pro g r a m s. 245 Mu l t i p l e 1614 Cell Cycle 20; Vol. 3 Issue 12

4 EVOLUTION OF NUCLEAR ENVELOPE AND NUCLEAR PORE op C h ig yr 4 00 t2 es nd La Bi c os protein sequence alignments were constructed using the T-Coffee246 MUSCLE247 programs. Phylogenetic analysis was performed using the maximum likelihood method as implemented in the PROTML program of the MOLPHY package.248 Most parsimonious evolutionary scenarios given a particular tree topology were reconstructed using the Dollop program of the Phylip package249 as previously described.214 COMPARATIVE GENOMICS OF THE NPC Domain Organization of the NPC Components. Figure 1 schematically shows the domain architectures of the structural subunits of the NPC proteins associated with its function. A feature e nc ie Figure 2. Phyletic distribution of the nuclear pore complex nuclear envelope components in eukaryotes. Species name abbreviations: Anopheles gambiae (Ag), Arabidopsis thaliana (At), Aspergillus nidulans (An), Caenorhabditis elegans (Ce), Cida albicans (Ca), Ciona intestinales (Ci), Cryptosporidium parva (Cp), Cyanidioschyzon merolae (Cm), Dictyostelium discoideum (Dd), Drosophila melanogaster (Dm), Encephalitozoon cuniculi (Ec), Danio rerio (Dr), Entam oeba histolytica (Eh), Giardia lamblia (Gl), Gibberella zeae (Gz), Guillardia theta (Gt), Homo sapiens (Hs). Leishmania major ((Lm (Lm), Lm), ), Magna porthe grisea (Mg), Mus musculus (Mm), Neurospora crassa (Nc), Oryza sativa (Os), Plasmodium falciparum (Pf), Plasmodium yoelii ((Py (Py), (Rn), Py), Rattus norvegicus Py), nor Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Takifugu rubripes (Fr). that immediately becomes apparent is the relative simplicity of the organization of NPC structural uctural components, with a marked prepreponderance of repetitive domains which are, typically,, involved in superstructure formation. The principal building blocks of the nucleoporins are WD40 (β-p ropeller) repeats, various forms of simple FG (phenylalanine-glycine) repeats, coiled-coil domains. Similarly, the karyopherins consist, to a large extent, of α-helical HEAT/ARM repeats. Furthermore, FG-repeats other simple repetitive structures are characteristic also of some of the Poms, whereas components of the Ran cycle contain LRR (leucine-rich repeats) in RanGAP RCC1 repeats in RanGEF (Fig. 1). It Cell Cycle 1615

5 appears that this remarkable abundance of repetitive domains is directly relevant for the formation of the symmetrical structure of the NPC; it also has implications for the origin evolution of the NPC as discussed in the subsequent sections of this article. The repertoire of nonrepetitive, globular domains in NPC proteins is small but these are of crucial importance for the NPC function, such as Ran GTPase domain, the RAN-binding domains, which have Cthe Ca a PH-d o m a i n-like fold, the NTF2 domain (see Fig. 2). shows the phyletic patterns (i.e., patterns of presence-absence in the sequenced genomes) of the NPC NE components (see additional details in SupplementaryTable opytable 1). The NPC Structure Evolutionary Conservation of NPC Components. The basal structure of the NPC is formed by the Nups. The pn Nups that are conserved throughout the eukaryotic kingdom are Gle2, y, Nup98 (Nup145N), Nup96 (Nup145C), Nup170 (Nup155), Sec13, Mad2, Nsp1 (Nup62) Nup53; the only exception is the Guillardia lardia ghnucleom theta nucleomorph which does not seem to encode any Nups (Fig. 2) The nucleomorph has a highly reduced genome seems to have lost the great majority of the genes that are conserved in other eukaryotes. 56,57 Since 20 electron-microscopic analysis indicates that the nucleomorph retains nuclear pores, 58 it appears most likely that, in this case, the Nups are scavenged from the host. In the rest of the eukaryotes, a relatively small number of Nups are ubiquitous, with the highly reduced genome of the m i c rosporidian parasite En c e p h a l i t o zoon cuniculi 5 9 being part i c u l a rly limited in this regard. This observation raises the question whether these components can form a minimal NPC. To address this this question properly, we must consider the NPC in terms of its functional architecture. Lchi- The Pore Membrane Domain the Cytoplasmic Nuclear yh nents.py larig rirdg to form a helical bundle with three bi-helical hairpin units has a conserved cysteine several highly conserved charged residues. This domain of Ndc1p is likely to mediate interactions with other conserved NPC components on the lumen surface. Pom152 is nonessential in yeast, while nothing, except for its localization to the NPC, is known about Pom34. 6,64 Synthetic lethal screens did, however, identified Nup170 (Nup155) the related Nup157 as interacting partners of Pom152 suggested that Nup170 is part of the nucleoplasmic cytoplasmic rings. 65 It has been shown that Nup53 (Nup35) interacts with membranes is i n vo l ved in the early stages of NPC assembly, pro b a b l y, by re c ru i ting Pom152 Ndc1p to the assembly site. 66 Nup170 is conserved in all eukaryotes Nup53 is present in the crown group G. lam - blia. Thus, both Nup170 Nup53 can be inferred to have been present in LECA, where they would have been involved in NPC assembly nuclear envelope interactions. Nup170 Nup53 also form a sub-complex with Nup59, which is a paralog of Nup53 appears to represent a lineage-specific duplication in fungi. 67 In addition, synthetic lethal screens indicate genetic interactions between Pom152, Nup188, Nic Nup188 is nonessential but interacts physically with Pom152 with Nic96 (Nup93) is symmetrically distributed in the central core of the NPC. 68 Nup188 Nic96 also interact with Nup192 (Nup205) Nup188, Nic96, Nup192 are limited to the crown group probably overlap in function with Pom152, just as Nup170. While two major integral pore membrane proteins, Ndc1p ionales nde s pbioscience ien ce GP210, are traceable to early stages of eukaryotic evolution, none of them is detected in the available sequence from G. lamblia. T h i s Lt s u ggests that LECA did not have integral membrane proteins in the NPC (see also below), although it cannot be ruled out that the Rings. The pore membrane domain is defined by the fusion Lahe of the anlambli G. lamblia orthologs of these proteins are highly divergent or were outer inner nuclear envelopes; embedded in this domain are the missing in the available data. Barring the latter possibility, it appears integral pore membrane proteins (Poms). The Poms are involved in that GP210 Ndc1p emerged in the common ancestor of the the initiation of pore complex formation stabilization, crown group or, in the case of the former, in the common ancestor f u n ction as the anchor site for other NPC components. 6,8 Pore of the clade that includes the crown group the Apicomplexans. membrane proteins so far identified are Pom34, Pom152, The presence dence e of the two types of Ig-domains, which otherwise cooccur Ndc1p in yeast, Pom121 the dimeric glycoprotein GP in a similar configuration only in bacterial cell surface adhesion ( a closely related paralog) in vertebrates. Despite their critical m o lecules, points to the origin of GP210 from a bacterial source function, not all Poms are widely conserved (Fig. 2). Pom34 through HGT. In phylogenetic trees, type-2 Ig domains of GP210 Pom152 are restricted to Fungi, whereas Pom121 is seen only in a re nested within the bacterial radiation of such domains, which ve rtebrates. Our analysis showed that Ndc1 GP210 (NUP210) f u rther supports a bacterial origin for this POM (See Fig. S1B a re present throughout the crown group (a term we use here to S2C). Considering that the inner outer NE membranes form a d e signate animals, fungi, plants, related groups of protists; see continuous surface, peripheral oprotei proteins, such as Nup170 Nup53, also below),, additionally, GP210 is also present in the could have been sufficient for membrane anchoring of the NPC. Apicomplexa. GP210 appears to have been secondarily lost in the Indeed, overexpression of Nup53 led to relocation of this protein to Fungi (Fig. 2 Fig. S1B C). Sequence profile analysis showed the nucleus formation of intranuclear ar tubular membranes which that GP210 shares a series of specialized immunoglobulin (Ig) fold progressed to double membrane lamellae e similar to the nuclear domains with the prokaryotic intimin-related cell-surface adhesion membrane. 66 Moreover, these membranes were interrupted by pores proteins. The bacterial intimin-related proteins contain two types of which, however, lacked NPC. Nup170 is involved ved in chromosome Ig domains (type-1-2) typified, respectively, by the N-terminal s e g regation has been implicated in interaction with chro omal C-terminal Ig domains of E. coli intimin, 61 both these types s u b d o m a i n s. 72 cee m o s C o n c e i va b l y, this could have been the original function of Ig domains are found in GP210. In most eukaryotes, GP210 of Nup170; subsequently, this protein, along with Nup53, might c o ntains five type-1 at least two type-2 Ig domains followed by have been recruited for the formation of the NE membrane e its a C-terminal membrane-spanning helix. By analogy to their bacterial attachment to chromatin. This scheme points to an intimate eo i n vo l vement of at least some of the highly conserved NPC components in the h o m o l o g s, 61 we predict that the Ig domains of GP210, which project into the lumen between the inner outer membrane, might evolution of the nucleus itself, perhaps to such an extent that they p rovide a surface for adhesive interactions involved in the assembly p l a yed a major role in the emergence of the nuclear stru c t u r a l of the NPC complex. Ndc1p, which is also involved in anchoring o r g anization. The subsequent emergence of Ndc1p in the crown the mitotic spindle pole, 62,63 is a 7-TM protein with a conserved, group could have strengthened the links between mitotic spindle large C-terminal globular domain (Fig. S1C). This domain is predicted formation the assembly of the membrane. This might have 1616 Cell Cycle 20; Vol. 3 Issue 12

6 played a role in the emergence of asymmetric cell division that is central to cell differentiation in several clades of the crown group. Cytoplasmic Fi l a m e n t s. The cytoplasmic filaments serve as d o c king ports of entry to the nuclear pore. Proteins that localize exclusively to the cytoplasmic side of the NPC in yeast include Nup159 (Nup214), Nup42, Nup82 (Nup88, 84). 6 Nup159 its vertebrate ortholog Nup214 are both localized to the cyto- Cplasmic filaments contain FXFG repeats, which suggests that docking of transport factors is the primary function of these repeats characteristic of several Nups. 73,74 Nup159/214 is restricted to the metazoan/fungi clade, whereas the FG-repeat-containing protein Nup42 o42 appears ars to be Sa c c h a ro m yc e s-specific. Nup82 (Nup88) is c o nserved ed pyown in the crown group lineages functions by anchoring filament proteins, including Nup159 (Nup214), Nup116 in yeast, Nup98 in ve rtebrates, rye ryrt tes, rtey Nsp1 (Nup62), RanBP2, to the NPC75-80 complex Nup98 is a nearly universal eukaryotic protein, Nsp1 is limited to the rihe crown gro u p, whereas RanBP2 is animal-s p e- cific (Fig. 2). Nup116 Nup100 ghare found only in Sa c c h a ro m yc e s; these p roteins contain an N-terminal GLFG repeat domain a distinct C-terminal autopeptidase domain which is also present in the universal nucleoporin Nup98 (Nup145N). Thus, Nup116 Nup100 apparently evolved in yeast as a result of lineage-specific duplications of Nup98. Nup116 not only serves as a docking site via its GLFG repeats, but also has a Gle2p-binding sequence (GLEBS) which anchors Gle2p at the nuclear pore. 81 Gle2p is a ~350 amino acid residue protein containing four WD40 repeats, hich toles which h is nearly u n iversally conserved in eukaryotes (Fig. 2), in addition ion to being a structural component of the NPC, is involved in RNA export. Leing Lrt. 82,83 Interestingly, yeast Nup145N does not contain a GLEBS LaNup2 motif, which is present in vertebrate Nup98. This motif has been shown Lato interact with Nup88 RAE1, the vertebrate ortholog of Gle2. 84 Thus, in yeast, duplication of Nup145N was probably followed by sub-functionalization. This is supported by the differences in the localization of Nup100 Nup145N: the former is found largely on the cytoplasmic side of the NPC the latter on the nucleoplasmic side. 6,85 The three paralogous nucleoporins show tri-partite localization, with Nup116 present in the cytoplasmic filaments, Nup100 at the cytoplasmic side of the Nup84 complex, Nup145N at the nucleoplasmic side of the Nup84 complex. 6 In contrast, vertebrate Nup98 is found in a complex with Nup88 in the cytoplasmic filaments with the Nup107 (yeast Nup84) complex located within the central core of the NPC, where it interacts with Nup96 on both sides of the NE plane. 78 This case illustrates modification of the NPC organization instigated by lineage-specific duplications of genes coding for core NPC components. Interaction of Gle2p with Nup98 the nearly universal presence of each of these proteins among eukaryotes suggest that they interacted in LECA, with Gle2p invo l ved in the maintenance of the NPC stru cture already at that stage of eukaryotic evolution. Nup98 Nup96 (Nu p 145 N-Nup145C) are a pair of interacting nucleoporins which, in animals, fungi, slime molds, C. parvum, a re encoded as a single fusion polypeptide. This Nu p 98-Nu p 96 p rotein is autocatalytically cleaved at a conserved HF S (or H[FY] T in some fungi) motif contained in the C-terminus of autopeptidase domain that adopts an unusual β-prism fold In plants, Nup98 Nup96 are encoded in separate genes. However, plant Nup98 proteins (or, in some cases, Nup96) typically retain the HFS motif. Moreover, E. histolytica E. cuniculi lack Nup96 but have Nup98 which ends at HFS or HF, re s p e c t i ve l y. While G. lamblia has a htch20 e Lan ndes d i ve rgent homolog of Nup96 (gi: ), it is not clear if it is a functional equivalent of the Nup96 of other eukaryotes, furthermore, the Nup98 proteins of Giardia Leishmania lack the HFS motif. These observations suggest that the Nup98-Nup96 fusion the emergence of the autocatalytic cleavage mechanism predate the divergence of the crown group lineages but might have not yet have evolved in LECA, although the latter conclusion hinges on the adopted phylogeny of eukaryotes (see below). Nup96 contains a conserved, large, α-helical domain that is also found in other Nups, such as Nup107 (yeast Nup84), the Sec31p (Web1p) protein family consisting of subunits of the COPII coat, which envelopes vesicles involved in transport from ER to Golgi. 90,91 Sec31p, which can be traced back to LECA, contains the Nup96-like domain to the C-terminus of a β-propeller structure formed by WD40 repeats. The Nup96-like domains are likely to form α -superhelix structures involved in docking of interacting proteins. The presence of a shared Nu p 96-like domain in the NPC proteins the vesicular coat protein Sec31p suggests a common origin for the structural components of the NPC ER-derived vesicular coats. The Nuclear Ba s k e t. Nup98 (Nup145N) contains GLFG repeats, 92 whereas Nsp1 (Nup62) has FXFG repeats. 93,94 Nup98 Nsp1 seem to have multiple roles in the NPC, being present in the cytoplasmic filaments, the central pore, the nuclear basket ring. 78,95 Since these nucleoporins are nearly universal (Fig. 2), it appears most likely that both types of FG repeats were present in LECA were already affixed to specific parts of the NPC where they facilitate docking transport. Many other nucleoporins also have been localized to the nuclear basket. These include Tpr (Mlp2), 96,97 Nic96 (Nup93), which is attached to Mlp2/Tpr, 96,98 Nup2 (Nup50), which also associates with the cytoplasmic fila- ments, 99,100 Nup153 (Nup1), 6,101 Nup60. 6 Linked to the nuclear basket is the nuclear ring that interacts with the rest of the ndther NPC. Other proteins localized to this part of the nuclear basket are Nup192 (Nup205), 70,71 Nup188, a paralog of Nup192 restricted to coelomate animals, 68,71 Nic96 (Nup93). 96,98 Tpr, Nic96, Nup192 are conserved ed in the eukaryotic crown group, whereas Nup1 Nup2 are limited to the Metazoan/Fungi lineage, Nup60 seems to be unique ue to S. cere v i s i a e ( Fig. 2). The phyletic d i stribution of the nuclear Blear basket components (Fig. 2). suggests that the minimal functional nal structure consists of Nup98 Nsp1 which, together with Gle2p, might have been the only components of the nuclear basket in LECA. The Central Pore. The central part of the NPC consists of the cytoplasmic nucleoplasmic rings, core components which a re composed of several distinct sub-c o m p l exes, including Nu p 170-Nu p 59-Nup53, Nu p 188-Po m 1 52-Ni c 96-Nup192, Nsp1-Nic96-Nup57-Nup49 referred to above. Among the subunits of these complexes, only Nup170 Nsp1 apparently were e present in LECA (Fig. 2). The only remaining sub-complex is the Nup84 (Nup107) complex which consists of Nup84 (Nup107), Nup145C (Nup96), Nup145N (Nup98), Sec13, Nup120 (Nup160), ceup85, Nup85, Seh1 Nup Nup98, Nup96, Sec13 are present in Bio iosc scie cien enc nce nbioscience e ost of cee all eukaryotes can be assigned to LECA, whereas the other s u bunits of the Nup84 complex are restricted to the crown group. The Ran Cycle. Proteins involved in the Ran-cycle machinery comprise the most highly conserved unit among all NPC components. Since Ran, RanGAP, NTF2, RanBP1, RanGEF are all required for efficient execution of the Ran cycle, 106,107 it is not unexpected that, with a few exceptions, such as the absence of RanGAP in s e veral protists, these proteins are nearly ubiquitous in eukaryotes Cell Cycle 1617

7 Cop opy pyri yrig righ ght Les Figure 3. Phylogenetic tree of the karyopherins. The maximum-likelihood tree was constructed as described in the text. The bootstrap support for individual nodes (>80%) is shown by circles. (Fig. 2). Notably, RanBP1 is missing in insects nematodes. Howe ve r, these organisms encode RanBP2, an animal-s p e c i f i c nucleoporin that has four RanBP1 domains. 108 It seems likely that RanBP2 functionally replaces RanBP1 in those organisms that have lost the latter. In addition to their role in the regulation of nuclear transport, Ran associated proteins are also involved in NPC NE assembly as well as spindle formation, it has been shown that these processes depend on GTP hydrolysis GDP-GTP exchange on Ran Thus, it appears likely that most of the Ran-cycle system was already in place before a complete NPC evolved that functions of Ran in chromosome segregation nuclear assembly predate those in nucleocytoplasmic transport. It is of interest that nuclear import of Ran is mediated by a distinct t r a n sport factor, NTF2, rather than a dedicated karyopherin. 113 A possible explanation for this might be that the NTF2-facilitated Ran transport evolved before the karyopherin system was in place. The Ran protein is distinguished from all other eukaryotic GTPases of the Ras-like superfamily by the presence of a conserved pro l i n e f o llowed by an acidic tail at the extreme C-terminus, in contrast to conserved cysteines which undergo fatty acid modification in most of the other Ras-like GTPases. 114 Thus, a major aspect of the divergence of the nuclear cytoplasmic membrane-associated Ras-like GTPases apparently was the acquisition of different C-terminal intracellular targeting signals. The Karyopherins. In terms of phyletic patterns, the karyopherins can be divided into several classes (Fig. 2). The first class, which includes Kap95, Kap60, Kap1, Kap121, Kap119, Kap124, Kap109, Kap127, Kap120 snurportin1, is conserved widely the eukaryotic kingdom, with some lacunae. The second class is limited to the crown group includes Kap111, Kap114, Kap122, Kap123 Kap142. T h e third class RanBP16 exportin 4 is present in animals plants but absent (probably lost) in Fungi. Finally, a number of karyopherins are lineagespecific, such as Kap108 (ye a s t s ), RanBP20 (animals), RanBP6, RanBP8, RanBP17 (vertebrates). The three t ruly ubiquitous karyopherins are Kap95p (importin β1), Kap60p (importin α), Kap109p (Cse1p) (Fig. 2). Notably, like the Ran cycle machinery, importins α β have additional functions in spindle formation NE NPC assembly, re s p e c t i ve l y It seems likely that these karyopherins comprised the primord i a l nuclear transport machinery, with importin α evolving cargo recognition capabilities to bind all cargo proteins containing a basic NLS, importin β1 evolving the capacity to recognize NPC components, in part i c u l a r, Kap109p, which is required for recycling of importin α. 118,119 Phylogenetic analysis of karyopherins reveals at least 13 distinct, well supported lineages re presented in animals, fungi, plants; of these, 6 7 were represented also in alveolates trypanosomes, 4 in Giardia (Fig. 3). Notably, the Giardia karyopherins branched off at the base of each subtree (data not shown), which is compatible with the possibility that they represent ancestral forms. Thus, evolution of this class of NPC proteins apparently involved early, stepwise duplications which were followed by additional, lineage-specific Bduplicati c expansion. This propagation of paralogous karyopherins resulted in increased specificity versatility of nuclear transport. More specifically, grouping g of importin β (Kap95) with Kap121 Kap1 in a clade suggests that these were e the first karyopherins to diverge evolve distinct import specificities. This is supported by their distribution throughout the eukaryotic kingdom, with only a small number of apparent losses (Fig. 2). Kap121 is involved in import of ribosomal proteins in vertebrates 120 enarious various proteins in yeast, including histones H3 H4, 121 the nct meiotic protein Sp o 1, 122 the transcription factors Pdr1, Yap1, Pho4 St e In addition, Kap121 has been implicated in mrna export 128 in nuclear pore assembly via binding of Nup53p. 129 The latter observation suggests that the duplication of the ancestral Kap95-like celike gene might predate the emergence of the complete NPC. Kap1 ekar (karyopherin β2b) mediates import of mrna-binding proteins r i b osomal proteins is, additionally, involved in mrna export via interaction with TAP, an mrna exporter that can also function independently of Kap Most of the TAP/Mex67 mrna export system components are found only in fungi animals (Fig. 2; see next section). Thus, Kap1 Kap121 might have Bioscience ien ce 1618 Cell Cycle 20; Vol. 3 Issue 12

8 been among the first proteins recruited for active mrna export. The only other readily identifiable relationship between nonorthologous karyopherins is that between Kap109 Kap119 which form a distinct clade apparently represent an ancient duplication ( Fig. 3). Like Kap109, Kap119 is found in nearly all eukaryo t e s ( Fig. 2). Kap109 is involved in the export of importin α (Kap60), 134, whereas Kap119 participates in the import of histones Ctranscription elongation factor IIS. 136 Thus, specialization of at least some of the karyopherin functions seems to predate the radiation of the currently known eukaryotic lineages, whereas much more extensive specialization occurred upon the advent of the crown group. Both sequence- structure similarity based clustering suggest that the closest pyes relatives of the karyopherins superfamily are the HEAT repeat proteins α β adaptin (data not shown), which are involved in linking coatamers to vesicles in cytoplasmic vesicular t r a n s p o rt. This yrrelations relationship suggests that yet another set of key c o mponents of vesicular transport in the ER nuclear pore on the nuclear membrane might have had a common origin. The two N-terminal ARM/HEAT AT repeats of the karyopherins comprise a conserved module, each ARM/HEAT unit in this module contains an additional helical insert between the h a i r p i n-forming helices. These karyopherin 2cal 207 rin N-terminal (K-N) m o dules (often referred to as IBB or IBN domains) are involved in distinctive interactions of these proteins. 20Snurportin1, 137 S which is involved in the m3g-cap-dependent nuclear import of spliceosomal U snrnps, also has a K-N module similar to that tin- of importin-α but differs from all other karyopherins in possessing a 4-α 4α C-terminal m3g-cap-binding domain. 138 An analysis of the m3g-cap-binding -bi domain using PSI-BLAST searches showed that it is homologous Ling ous to the guanylyl transferase domain of the capping enzyme. While Lanents the homolog of the cap-binding domain in snurportin1 contains all the critical charged residues invo l ved in polar interactions with the s u bstrate that are conserved in this family, it lacks the N-terminal catalytic lysine aspartate, which directly participate in nucleotidyl transfer in the capping enzymes related nucleic acid ligases 139 (see the alignment in Fig. S1A). This observation suggests that the guanylyl transferase domain of snurportin1 is catalytically inactive; however, since it retains the substrate-binding residues, it is likely to bind the 5 cap structures similarly to the active enzyme (Fig. S1A). Thus, snurportin1 resembles the eukaryotic CG6379 family of FtsJ/RrmJ-like RNA methylases, which also possess a cap-binding domain derived from an inactive guanylyl transferase domain of the capping enzyme. 140 Snurportin1 is found in animals, plants, the apicomplexan Cryptosporidium parv u m but apparently was lost in fungi. It seems likely that this unique karyopherin was derived just prior to the radiation of the crown group, via the fusion of a paralog of the guanylyl transferase domain of the capping enzyme with a K-N module derived from importin-α. The TAP/Mex67 mrna Export System. Transport of mrnas is much more complex than protein transport in terms of the multitude of players identified (Figs. 1 2). The central components of this system, TAP/Mex67 Mtr2/NXT1, are found only in Fungi Me t a zoa, which suggests a re l a t i vely late origin of the entire s y stem. Those components of this system that are conserve d throughout the eukaryotic kingdom all have other functions, such as involvement in mrna splicing (Yra1, Sub2p) 141,142,143 transcription (Hpr1, Tho2, Thp1, Thp2, Sus2, Sac3, Na b 2 p ). 34,35, Therefore, it appears most likely that these proteins evolved primarily for their mrna processing capacities were subsequently coopted for mrna export at the base of the t k f u ngal-metazoan clade, to supplant the ancestral passive mrna export with an ATP-dependent, regulated mechanism. Plants have a lineage-specific derivation of the NTF2 family (e.g., the At5g440 protein in Arabidopsis), in which the NTF2 domain is fused to a MAP kinase domain. This protein is likely to be involved in a plant-specific nuclear transport system similarly to fungal Mtr2p. 148 The Ribosomal Subunit Export (RIX) System. The mechanisms of export of the 60S 40S ribosomal subunits are not yet understood in detail, but the study of the RIX mutants proteomic analysis of factors associated with nuclear 60S subunits have revealed the notable potential complexity of this nuclear export system. In yeast, two paralogous GTPases, Nug1p Nug2p, have been identified as key components of the ribosomal subunit export apparatus in both genetic physical interaction screens. 149 These proteins contain a circularly permuted GTPase domain of the YjeQ-YqlF family which is widely represented in bacteria, archaea eukaryotes. 150,151 Phylogenetic analysis of this family of GTPases reveals a primary split between the bacterial the archaeo-eukaryotic lineages (supported by specific sequence motifs shared by the archaeoeukaryotic branch), which is best compatible with the presence of this GTPase in the last universal common ancestor of modern life forms (ref. 150; see the phylogenetic tree in Fig. S2A). Several lines of evidence suggest that bacterial GTPases of the YjeQ family are involved in translation regulation, probably, in ribosomal assembly. 152,153 All eukaryotes have at least two paralogous GTPases of this family, Nug1p/Nug2p Nog2p, which suggests a duplication at the onset of eukaryotic evolution, prior to the time of LECA. Nog2p is invo l ved in the assembly of the 60S ribosomal subunit, 154 w h e reas the Nug1p/Nug2p proteins interact with the NPC RIX compo- nents are re q u i red for ribosomal export. 149 Thus, the duplication of the permuted GTPase in eukaryotes appears to correlate with the emergence of the nuclear compartment, with the Nug1/2p lineage acquiring the new role of coupling ribosomal transport assembly. Two deu other proteins that can be traced back to LECA are associated with Nu g 1 p / Nu g 2 p, namely, the AAA+ AT Pase midasin, a paralog of the cytoplasmic c dynein 155, 156 ), the BRC T-d o m a i n-c o n t a i ning protein pescadillo/nop7p. op7p 157,158 The other conserved components of the ribosomal export system are the Noc proteins (Noc1-4p), which contain specialized HEAT repeats 159 are likely to be functionally analogous to HEAT-repeat-containing -c karyopherins. Noc1p Bd BA (MAK21p), Noc2p, Noc4p are present in the crown group eukaryotes, apicomplexans, Giardia,, suggesting that each of these proteins was already represented resented in LECA. In contrast, Noc3p seems to be restricted to the crown group apicomplexans. Thus, the original core of the ribosomal osxport export system in LECA appare n tly consisted of the permuted GTPase linking assembly export of the ribosomal subunits at least three e specialized HEAT repeat proteins of the NOC group.two additional components of the ribosomal subunit export system, the CDC48p-like AAA+ AA+ ncc AT Pase Rix7p 160 the fast-evolving α-helical protein Rix1p, 149 are detectable only in the crown group lineages probably we re re c ruited for to f u n ction in ribosomal export later than the core components. 20 biles s eciabioscience ios cien ence e COMPARATIVE GENOMICS AND EVOLUTION OF THE NE AND THE ASSOCIATED NUCLEOSKELETON The nuclear envelope the underlying lamina have been s t u died in greatest detail in animals, especially in the context of human genetic diseases caused by mutations of the respective genes known as laminopathies. 54,161 The proteins associated with Cell Cycle 1619

9 EVOLUTION OF NUCLEAR ENVELOPE AND NUCLEAR PORE op C h ig yr 4 00 t2 La es nd Figure 4. Multiple sequence alignment of the Ig-fold fold domain of the lamin tails (LTD). Multiple sequence alignment of the LLTD domains was constructed using the T-Coffee program after parsing high-scoring pairs from PSI-BLAST secondary structure predicted using the JPRED program is shown BLAST search results. The secondar above the alignment with H representing an α-helix -helix E representing a _ str. The consensus shown below the alignment was derived using the following amino acid classes: hydrophobic (h: ALICVMYFW); aliphatic subset of the hydrophobic class (l; ALIVMC); the aromatic subset of the hydrophobic class (a; FHWY); alcohol (o: ST); small (s: ACDGNPSTV); the tiny subclass of small (u; GAS); polar (p: CDEHKNQRST); negative (-, ( DE); positive (+, HKR); big (b, KFILMQRWYE). The limits of the domains are indicated by the residue positions, on each end of the sequence. The numbers within the alignment are nonconserved inserts ts that have not been shown. The sequences are denoted by their gene name followed by the species abbreviation GenBank nidulans, Avar: Avar: Anabaena variabilis Identifier (gi). The species abbreviations are Ana: Nostoc sp., Anid: Aspergillus nidulans, variabilis, Bfun: Burkholderia fungorum, aurantiacus, Ceff: Corynebacterium Cor Blon:Bifidobacterium longum, Bthe: Bacteroides thetaiotaomicron, Caur: Chloroflexus aurantiacus, efficiens, Cglu: Corynebacterium elegans Dm: Drosophila melanogaster,dhaf: glutamicum, Cper: Clostridium perfringens, Cthe: Clostridium therm o c e l l u m, Ce: Caenorhabditis elegans, violaceus Hs: Homo sapiens, Hsp: Desulfitobacterium hafniense, Drad: Deinococcus radiodurans, Exsp: Exiguobacterium sp.., Gvio: Gloeobacter violaceus, punctiforme, Oihe: Oceanobacillus iheyensis, Pae: Halobacterium sp., Mmaz: Methanosarcina mazei, Mdeg: Microbulbifer degradans, Npun: Nostoc punctiforme, aver mitilis Scoe: Streptomyces coelicolor, Pseudomonas aeruginosa, Pgin: Porphyromonas gingivalis, Rmet: Ralstonia metallidurans, Save: Streptomyces avermitilis avermitilis, Smel: Sinorhizobium meliloti, Smu: Saccharothrix mutabilis, Sone: Shewanella oneidensis, Ssp: Synechocystis sp. thermophilus, Vpar: Vibrio sp., Tthe: Thermus ther parahaemolyticus. Bi isolated without a lamina using nondissociating conditions. 85 Other characterized characte rized nucleoskeleton than the animal lamins,, the best characterized coi led-coil led-c oil proteins protei appare n tly components are from plants; these are coiled-c coiled-coil unrelated to lamins.172,173 Other coiled-coil oil proteins from from nonmetazoans, notably yeast Trypanosoma,, also have been implicated as nucleoskeleton components.85,174 Coiled-coil coil domains are are ideal for self-assembly, it appears likely that different ent proteins with this structure have been re c ruited for nucleoskeletal function multiple on on mult multi iple ple occasions during eukaryotic evolution. Using iterative database searches, we detected domains homologous ologous olo gous to the LTD in several uncharacterized proteins from om phylogeneti phylogenetically cally diverse bacteria two archaea, Methanosarcina Halobacterium (Fig. 4), but not in other eukaryotes. The protein Chlo1887 (gi: ) from Chloroflexus resembles the animal lamins in p o ssessing a C-terminal LTD with an N-terminal low-complexity region a coiled-coil region similar to those found in lamins. In several bacterial proteins, the LTD cooccurs with membrane-associated e nc Cell Cycle ie 1620 c os this structure are central to the interactions with chromatin maintenance of nuclear integrity. In animals, the lamina comprises an extensive protein meshwork which underlies the inner membrane of the NE forms numerous contacts with the proteins of the inner membrane the chromatin.162 The lamina is linked to the NE through numerous integral membrane proteins, the most abundant best characterized being Lap1, Lap2, emerin, Man1, lamin-b receptor (LBR), Unc-84/Sad-1, nesprin1, NUANCE Nuclear Lamins. Nuclear lamins are the primary structural components of the lamina. The lamins are intermediate filament p roteins with a long coiled-coil region followed by a globular C-terminal lamin-tail domain (LTD) which has the immunoglobulin (Ig) fold. 170 Intermediate filaments are involved in the maintenance of cellular integrity are abundant in cells that are subject to physical stress, such as muscle.171 Not much is known about nucleoskeletons in organisms other than animals. Yeast does not have a discernible lamina as indicated by the fact that the nucleus can be 20; Vol. 3 Issue 12