1.I.1 The Nuclear Pore Complex (NPC) Family [formerly 1.A.75]

Numerous Nuclear Pore Complex (NPC) proteins, called nucleoporins, have been identified and characterized from vertebrates and yeast (Brohawn et al., 2009; Beck and Hurt 2017). Thirty such proteins are recognized constituents of the yeast NPC, and at least 50 nucleopore proteins have been characterized from vertebrates. Many of these proteins have been tabulated for (1) S. cerevisiae and (2) vertebrates by Stoffler et al. (1999), Tran and Wente (2006) and Beck and Hurt (Beck and Hurt 2017). It is known that nuclear proteins contain short sequences called 'nuclear localization sequences' (NLS) that target them for nuclear import. A nuclear localization sequence receptor and several cytosolic factors appear to play roles in nuclear import of NLS-bearing proteins. Nuclear export signals (NESs) have also been identified. Several different forms of each type of targeting signal have been identified that lack homology to each other and may be recognized by different receptors. NLS and NES receptors (termed importins and exportins, respectively) may all be homologous and are members of the karyopherin-β/importin-βsuperfamily (Sorokin et al., 2008). The many protein constituents of NPCs have been discussed from structural, topological and functional standpoints by Panté and Aebi (1996), Nigg (1997) and Stoffler et al. (1999). Evidence for an involvement of the specific receptors or shuttling vectors, such as the importin-β-family member, Msn5 (spP52918), has been presented (Kaffman et al., 1998). Structural features and functional correlates have been discussed by Talcott and Moore (1999) as well as Gorlich and Kutay (Görlich and Kutay, 1999).The Nuclear Pore Complex (NPC) facilitates molecular trafficking between nucleus and cytoplasm and is an integral feature of the eukaryote cell, and LINC complexes can affect nucleo-cytoplasmic transport through the NPC (Jahed et al. 2016).  The NPC exhibits eight-fold rotational symmetry and is comprised of approximately 30 nucleoporins (Nups) in different stoichiometries. Nups are broadly conserved between yeast, vertebrates and plants, but few have been identified among other major eukaryotic groups. The nuclear pore consists of 12 subcompartments: (1) the cytoplasmic ring, (2) the inner pore ring, (3) the nuclear ring, (4) the peripheral elements, (5) the nuclear basket, (6) the cytoplasmic filament, (7) the Y complex, (8) the inner right complex, (9) the transmembrane NUPs, (10) the NUP62 complex (nucleoskeletal-like 1 (Nsp1), (11) the cytoplasmic complexes, and (12) the nuclear basket complex.  These are color coded, indicating the proteins included, both in humans and in yeast, by Beck and Hurt 2017. Nucleopores can dilate and constrict in cellulo because the NPC scaffold is mechanosensitive, and membranee tension regulates its diameter (Zimmerli et al. 2021). The complexity of the NPC has been discussed (Rush et al. 2023). The nuclear pore complex privides a platform for epigenetic regulation ().  The Nuclear Pore Complex (NPC) exhibits eight-fold rotational symmetry and is comprised of approximately 30 nucleoporins (Nups) in different stoichiometries. Nups are broadly conserved between yeast, vertebrates and plants. Neumann et al. (Neumann et al., 2010) screened for Nups across 60 eukaryote genomes and reported that 19 Nups (spanning all major protein subcomplexes) are found in all eukaryote supergroups studied (Opisthokonts, Amoebozoa, Viridiplantae, Chromalveolates and Excavates). Based on parsimony, between 23 and 26 of 31 Nups can be placed in the last eukaryotic common ancestor (LECA). Notably, they include central components of the anchoring system (Ndc1 and Gp210) indicating that the anchoring system did not evolve by convergence, as has previously been suggested. These results significantly extend earlier results and, importantly, unambiguously place a fully-fledged NPC in LECA. Vesicle coating complexes share a common evolutionary origin with Nups, and can be traced back to LECA. Surprisingly, only three supergroup-level differences (one gain and two losses) between the constituents of COPI, COPII and Clathrin complexes were formed. The results indicated that all major protein subcomplexes in the Nuclear Pore Complex are traceable to the Last Eukaryotic Common Ancestor (LECA), regardless of the position of the root of the eukaryotic tree (Neumann et al., 2010).  In humans, approximately 60 proteins are involved in nuclear transport, including the nucleoporins that form membrane-embedded nuclear pore complexes, karyopherins that transport cargoes through these complexes, and Ran system proteins that ensure directed and rapid transport. Selinexor (KPT-330), an inhibitor targeting the nuclear export factor XPO1 (also known as CRM1), was approved in 2019 to treat two types of blood cancers, and dozens of clinical trials of are ongoing (Yang et al. 2023). They summarized approximately three decades of research data in this field, focusing on the structure and function of individual nuclear transport proteins, providing a cutting-edge and holistic view on the role of nuclear transport proteins in health and disease.

The symmetric core of the nuclear pore complex can be considered as a series of concentric cylinders. A peripheral cylinder coating the pore membrane contains the elongated heptamer that harbors Sec13-Nup145C in its middle section. Sec13-Nup145C crystallizes as a hetero-octamer. Oligomerization of Sec13-Nup145C is due to numerous interacting surfaces in the hetero-octamer, which forms a slightly curved, yet rigid rod of sufficient length to span the entire height of the proposed membrane-adjacent cylinder. In concordance with the dimensions and symmetry of the nuclear pore complex core, Hsia et al. (2007) suggested that the cylinder is constructed of four antiparallel rings, each ring being composed of eight heptamers arranged in a head-to-tail fashion. This model suggests that the hetero-octamer vertically traverses and connects the four stacked rings. See Hsia et al., 2007 for a detailed picture of subcomplexes and thin arrangements in the NPC. Bilokapic and Schwartz (2012) have summarized the state of NPC structural efforts, described the breakthroughs of recent years, and pointed out the existing disputes in the field. Stuwe et al. 2015 have determined the crystal structure of the nuclear pore complex coat (~400 kilodaltons) from Saccharomyces cerevisiae at 7.4Å resolution.  More recently, the architecture of the symmetic core of the nuclear pore has been elucidated (Lin et al. 2016; Stuwe et al. 2015; Stuwe et al. 2015). Cell stretching modulates the characteristic time needed for the nuclear import of small inert molecules (García-González et al. 2018). The nuclear pore compex facilitates spatial organization and epigenetic regulation of the genome (Sump and Brickner 2022; Brickner 2023). 

Members of the importin-β family of transport receptors mediate NPC passage of cargo by interacting with nucleoporins and a small GTPase, Ran. Ran acts as a molecular switch by interconverting between a GTP and GDP binding state, regulated by a nuclear GTP/GDP exchange factor, RCC1, and a cytoplasmic GTPase-activating factor, RanGAP. The asymmetric distribution of these proteins insures that nuclear Ran is primarily in the GTP-bound form, but cytoplasmic Ran is in the GDP-bound form. This gradient of Ran-GTP ensures release of cargo from the transport importin-β receptors which bind NLS-substrate/importin-β complex in the cytoplasm, and this ternary complex dissociates by binding RanGTP to importin-β in the nucleus. While ATP (or GTP) is required for nuclear export of importin-β, it is not required for nuclear import. Smad2/Smad4 heterocomplexes, formed in the cytoplasm, are imported through the nuclear pore complex as entireties, and finally dissociate in the nucleus (Li et al. 2018).  Defects in the NPC can lead to diseases (Fare and Rothstein 2024).

Mediators of import into the nucleus (importins) and export mediators (exportins) interact with RanGTP but respond to the nucleocytoplasmic RanGTP gradient in diametrically opposed ways (Mingot et al., 2004). Importins bind cargo at low RanGTP levels in the cytoplasm and release cargo upon RanGTP binding in the nucleus. In contrast, exportins recruit cargo at high RanGTP concentrations, as ternary cargo/exportin/RanGTP complexes, in the nuclear compartment and release cargo when the Ran-bound GTP molecule is hydrolyzed in the cytoplasm. This active control of cargo binding and release by the RanGTPase system constitutes the sole input of metabolic energy into these transport cycles and is sufficient to allow importins and exportins to accumulate cargoes actively against gradients of chemical activity.  Highly variable molecular signatures of TDP-43 loss of function are associated with nuclear pore complex (NPC) injury in a population study of sporadic ALS patient iPSNs (Rothstein et al. 2023).

Transport through the NPC occurs by facilitated diffusion of the soluble carrier proteins or carrier-cargo complexes (Macara, 2001). Vectorality is provided by compartment-specific assembly and disassembly of the carrier-cargo complexes, often mediated by the Ran GTPase as noted above. The carriers recognize localization signals on the cargo and bind to pore proteins (Macara, 2001). While the yeast NPC is complex, those in plants and animals are much more so with hundreds of proteins functioning in various capacities. Many of the yeast NPC constituents can be found in other eukaryotes (e.g., vertebrate centrins function as does Cdc31p of yeast and plays a role in mRNA and protein export) (Resendes et al., 2008). The RNA U small nuclear (sn)RNA export adaptor protein, or the phosphorylated adaptor for RNA export, regulates U snRNA nuclear export to the cytoplasm in metazoa. It is phosphorylated in the nucleus and exported as part of the U snRNA export complex where it is dephosphorylated, causing complex disassembly (Kitao et al., 2008).

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) represent a notable exception. King et al. (2006) have shown that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-β and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-β, karyopherin-β1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. King et al. (2006) provided evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.

The transport receptor Mex67-Mtr2 functions in mRNA export, and also, using a loop-confined surface on the heterodimer, it binds to and exports pre-60S particles. Mex67-Mtr2, through the same surface that recruits pre-60S particles, interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains (Yao et al., 2007). In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Nup85 is the subunit in the Nup84 complex that binds to the Mex67 loop, an interaction that is crucial for mRNA export.

NPCs are proteinaceous assemblies of approximately 50 MDa of 456 known constituents that selectively transport cargoes across the nuclear envelope. Half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold (Alber et al., 2007). The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents. The most striking of these is a 16-fold repetition of 'columns'. How nucleoporins interact with the nuclear membrane and how this interaction contributes to NPC assembly, stability and function as well as shaping of the pore membrane has been reviewed (Hamed and Antonin 2021).

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Terry and Wente (2007) generated in S. cerevisiae a set of more minimal pore (mmp) mutants lacking specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. mRNA export also requires two NPC substructures-one on the nuclear NPC face and one in the NPC central core.

A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. Terry & Wente (2009) reviewed the structural features, cellular functions, and evolutionary conservation of the FG-Nups. The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) (Mudumbi et al. 2016).

Nups are among the few proteins linked to speciation from hybrid incompatibility in Drosophila (McQuarrie et al. 2023). Coding sequence evolution of Nup96 and Nup160 showed positive selection driving nucleoporin evolution. Channel Nup54 functionality is required for neuronal wiring underlying the female post-mating response induced by male-derived sex-peptide. A region of rapid evolution in the core promoter of Nup54 suggests a critical role for general transcriptional regulatory elements at the onset of speciation. Indel driven rapid evolution of core nuclear pore protein gene promoters has been ocumented Hence, the nuclear pore complex may act as a nexus for species-specific changes via nucleo-cytoplasmic transport regulated gene expression (McQuarrie et al. 2023).

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit in yeast utilizes three different receptors, Crm1, Mex67/Mtr2, and Arx1 which collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S ribosomal subunit export in higher eukaryotes. Several receptors can function in export. This helps explain how different export receptors could have evolved. Lo and Johnson (2009) have reviewed the structural features, cellular functions, and evolutionary conservation of the FG-Nups.

Nuclear transport receptors (NTRs) bind cargo molecules and supply nuclei with proteins and the cytoplasm with nuclear products like ribosomes. The facilitated mode of NPC passage reaches a capacity of up to 1,000 translocation events per NPC per second and accommodates objects of up to nearly 40 nm in diameter (Hülsmann et al., 2012). NTRs can utilize an energy input, e.g., from the RanGTPase system to accumulate substrates against steep concentration gradients.  

NPCs are built from ∼30 different nucleoporins (Nups) that can be classified into structural Nups and phenylalanine-glycine repeat-containing Nups (FG Nups). The structural Nups form the NPC scaffold and provide binding sites for the nonglobular FG Nups which are critical for the barrier. (Strawn et al., 2004; Frey and Görlich, 2007; Patel et al., 2007). An FG-Nup typically has hundreds of residues with up to 50 FG, FxFG, or GLFG motifs.

FG motifs bind NTRs during facilitated translocation and such interactions render NPCs 100- to >1,000-fold more permeable for NTR⋅cargo complexes than for inert objects of similar size. Molecules that are not bound to an NTR are blocked from NPC passage. Protein-protein interactions (PPIs) for three conserved proteins in the fission yeast, Schizosaccharomyces pombe, that localize to the inner nuclear membrane: Cut11/Ndc1 (O13961), Lem2 (O13681) and Ima1/Samp1/Net5 have been determined (Varberg et al. 2020).

The favored 'selective phase model' (Hülsmann et al., 2012) assumes that the barrier-forming FG domains comprise many cohesive units, which bind each other and thereby mediate multivalent interactions within and between individual FG domains. Such interactions result in a sieve-like FG hydrogel that allows passage of small molecules but suppresses fluxes of larger ones. NTRs overcome this size limit by binding to FG motifs and consequently disengaging FG meshes in their immediate vicinity. This way, NTRs can partition into the FG hydrogel and exit the barrier on the trans side (Hülsmann et al., 2012).

Nuclear export of mRNAs was thought to occur exclusively through nuclear pore complexes. However, Speese et al. (2012) identified an alternate pathway for mRNA export in muscle cells where ribonucleoprotein complexes involved in forming neuromuscular junctions transit the nuclear envelope by fusing with and budding through the nuclear membrane.

Rothballer and Kutay (2013) have discussed the biogenesis of NPCs during interphase of the cell cycle. This process requires a mechanistically enigmatic fusion step between the inner and the outer nuclear membrane. They focus on the principle of membrane pore formation in the nuclear envelope and consider existing paradigms of other cellular membrane remodeling events. The emerging roles of transmembrane proteins and membrane-shaping factors in NPC biogenesis are considered. Transport of integral proteins to the innwe nuclear membrane involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex (Ohba et al. 2004).

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements (Rothballer et al. 2013). LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have revealed the molecular basis of nucleo-cytoskeletal coupling (Rothballer et al., 2013). They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. 

The majority of nuclear import pathways are mediated by importin-cargo interactions, but not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. Lu et al. 2014 identify a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy, leading to nuclear accumulation. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway used by AR-containing transcriptional regulators (Lu et al. 2014). 

Stuwe et al. 2015 presented the reconstitution of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. They proposed that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation (Stuwe et al. 2015). 

NPCs mediate nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Chug et al. 2015 reported a structural analysis of the frog FG Nup62•58•54 complex. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. Chug et al. 2015 further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. Chug et al. 2015 suggested that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Ciliates have two functionally distinct nuclei, a transcriptionally active somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Iwamoto et al. 2015 showed, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Addition of new nuclear envelope (NE) precursors occured through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD resulted in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Thus, NPC type switching plays a crucial role in the establishment of nuclear differentiation in ciliates (Iwamoto et al. 2015). 

The NPCs in ciliates consists of about 30 different nucleoporins each. Iwamoto et al. 2017 presented evidence for compositionally distinct NPCs that form within a single cell of Tetrahymena thermophila. Each cell contains both a MAC and a MIC. Iwamoto et al. 2017 identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs, and the transmembrane components, Pom121 and Pom82, localize exclusively to MAC and MIC NPCs, respectively. Thus, functional nuclear dimorphism in ciliates depends on compositional and structural differences. Neurocystin (NPHP) proteins (e.g., O15259) anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle (Blasius et al. 2019).

A protein known to localize to and be important in the assembly of both the yeast NPC and the spindle pole body, which functions as the microtubule organizing center, is the 6 TMS protein, Ndc1p (NPC1 in humans). The N- and C-termini of Ndc1p are exposed to the cytoplasm (Lau et al. 2006). The paralogous Brr6 and Brl1 are conserved integral membrane proteins of the nuclear envelope (NE). Depletion of Brr6 and Brl1 caused defects in NPC biogenesis, whereas the already assembled NPCs remained unaffected. This NPC biogenesis defect was not accompanied by a change in lipid composition. However, Brl1 interacted with Ndc1 and Nup188 as well as transmembrane and outer and inner ring NPC components, indicating a direct role in NPC biogenesis.Both Brr6 and Brl1 associated with a subpopulation of NPCs and emerging NPC assembly sites. BRL1 overexpression affected NE morphology and suppressed the nuclear pore biogenesis defect of Δnup116 and Δgle2 cells. Possibly Brr6 and Brl1 transiently associate with NPC assembly sites where they promote NPC biogenesis (Zhang et al. 2018).

Importin-beta, Ran GTPase (Ran), RanGAP, and RanBP2 have been identified as proteins interaction with a thioredoxin-like protein, TMX2 (Oguro and Imaoka 2019). Importin-beta is an adaptor protein which imports cargoes from the cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-beta complex, which allows the recycling of importin-beta and reentry of Ran into the nucleus. Oguro and Imaoka 2019 showed that TMX2 is not a transport cargo. It localizes in the outer nuclear membrane with its N- and C-termini facing the cytoplasm, where it co-localizes with importin-beta and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and cysteine 112 of Ran is important in its regulation by TMX2. Knockdown of TMX2 suppressed importin-beta-mediated transport of proteins. Thus, TMX2 works as a regulator of protein nuclear transport, and it facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins (Oguro and Imaoka 2019).

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane. Mudumbi et al. 2020 distinguished protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains, containing nuclear localization signals, into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels without blocking their translocation using the peripheral channels. Blocking the peripheral channels blocks translocation through both channels (Mudumbi et al. 2020). In the June 10th 2022 issue of Science, a written symposium appeared focusing on structural aspects of the NPC. Emphasis was placed on the Cytoplasmic Ring. Several additional proteins were identified (Bley et al. 2022; Petrovic et al. 2022).

The bi-directional nucleocytoplasmic shuttling of macromolecules like molecular signals, transcription factors, regulatory proteins, and RNAs occurs exclusively through Nuclear Pore Complexes (NPC) residing in the nuclear membrane. This complex is essentially a congregation of ~32 conserved proteins termed Nucleoporins (Nups) present in multiple copies and mostly arranged as subcomplexes to constitute a functional NPC (Bindra and Mishra 2021). Nups participate in ancillary functions such as chromatin organization, transcriptional regulation, DNA damage repair, genome stabilization, and cell cycle control, in addition to their central role as nucleocytoplasmic conduits. Thus, Nups play a role in the maintenance of cellular homeostasis. In mammals, three nucleoporins that traverse the nuclear membrane are called transmembrane Nups (TM-Nups) and are involved in multiple cellular functions. They are NUP35 (also called MP44) of 326 aas with 1 or 2 possible central TMSs, NUP219 of 1887 aas and 2 TMSs, N- and C-terminal, and NUP121 (POM121) of 1249 aas with two N-terminal TMSs. Owing to their vital roles in cellular processes and homeostasis, dysregulation of nucleoporin function is implicated in various diseases such as cancer. Bindra and Mishra 2021 emphasize the distinct canonical and non-canonical functions of mammalian TM-Nups and the underlying mechanisms of their disease association.

The NPC is structurally conserved across eukaryotes as are many of the pore's constituent proteins. The transmembrane nuclear pore proteins, GP210 and NDC1, span the nuclear envelope holding,,the nuclear pore in place. Orthologues of GP210 and NDC1 in Arabidopsis were investigated through characterisation of T-DNA insertional mutants. While the T-DNA insert into GP210 reduced expression of the gene, the insert in the NDC1 gene resulted in increased expression in both the ndc1 mutant as well as the ndc1/gp210 double mutant (Collins et al. 2023). The ndc1 and gp210 individual mutants showed little phenotypic difference from wild-type plants, but the ndc1/gp210 mutant showed a range of phenotypic effects. As with many plant nuclear pore protein mutants, these effects included non-nuclear phenotypes such as reduced pollen viability, reduced growth and glabrous leaves in mature plants. The ndc1/gp210 double mutant exhibited nuclear-specific effects including modifications to nuclear shape in different cell types. Functional changes to nuclear transport in ndc1/gp210 plants were observed. The lack of phenotypes in individual insertional lines, and the relatively mild phenotype suggest that additional transmembrane nucleoporins, such as the recently-discovered CPR5, likely compensate for their loss (2006) provided evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.

The transport receptor Mex67-Mtr2 functions in mRNA export, and also, using a loop-confined surface on the heterodimer, it binds to and exports pre-60S particles. Mex67-Mtr2, through the same surface that recruits pre-60S particles, interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains (Yao et al., 2007). In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Nup85 is the subunit in the Nup84 complex that binds to the Mex67 loop, an interaction that is crucial for mRNA export.

NPCs are proteinaceous assemblies of approximately 50 MDa of 456 known constituents that selectively transport cargoes across the nuclear envelope. Half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold (Alber et al., 2007). The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents. The most striking of these is a 16-fold repetition of 'columns'. How nucleoporins interact with the nuclear membrane and how this interaction contributes to NPC assembly, stability and function as well as shaping of the pore membrane has been reviewed (Hamed and Antonin 2021).

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Terry and Wente (2007) generated in S. cerevisiae a set of more minimal pore (mmp) mutants lacking specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. mRNA export also requires two NPC substructures-one on the nuclear NPC face and one in the NPC central core.

A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. Terry & Wente (2009) reviewed the structural features, cellular functions, and evolutionary conservation of the FG-Nups. The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) (Mudumbi et al. 2016).

Nups are among the few proteins linked to speciation from hybrid incompatibility in Drosophila (McQuarrie et al. 2023). Coding sequence evolution of Nup96 and Nup160 showed positive selection driving nucleoporin evolution. Channel Nup54 functionality is required for neuronal wiring underlying the female post-mating response induced by male-derived sex-peptide. A region of rapid evolution in the core promoter of Nup54 suggests a critical role for general transcriptional regulatory elements at the onset of speciation. Indel driven rapid evolution of core nuclear pore protein gene promoters has been ocumented Hence, the nuclear pore complex may act as a nexus for species-specific changes via nucleo-cytoplasmic transport regulated gene expression (McQuarrie et al. 2023).

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit in yeast utilizes three different receptors, Crm1, Mex67/Mtr2, and Arx1 which collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S ribosomal subunit export in higher eukaryotes. Several receptors can function in export. This helps explain how different export receptors could have evolved. Lo and Johnson (2009) have reviewed the structural features, cellular functions, and evolutionary conservation of the FG-Nups.

Nuclear transport receptors (NTRs) bind cargo molecules and supply nuclei with proteins and the cytoplasm with nuclear products like ribosomes. The facilitated mode of NPC passage reaches a capacity of up to 1,000 translocation events per NPC per second and accommodates objects of up to nearly 40 nm in diameter (Hülsmann et al., 2012). NTRs can utilize an energy input, e.g., from the RanGTPase system to accumulate substrates against steep concentration gradients.  

NPCs are built from ∼30 different nucleoporins (Nups) that can be classified into structural Nups and phenylalanine-glycine repeat-containing Nups (FG Nups). The structural Nups form the NPC scaffold and provide binding sites for the nonglobular FG Nups which are critical for the barrier. (Strawn et al., 2004; Frey and Görlich, 2007; Patel et al., 2007). An FG-Nup typically has hundreds of residues with up to 50 FG, FxFG, or GLFG motifs.

FG motifs bind NTRs during facilitated translocation and such interactions render NPCs 100- to >1,000-fold more permeable for NTR⋅cargo complexes than for inert objects of similar size. Molecules that are not bound to an NTR are blocked from NPC passage. Protein-protein interactions (PPIs) for three conserved proteins in the fission yeast, Schizosaccharomyces pombe, that localize to the inner nuclear membrane: Cut11/Ndc1 (O13961), Lem2 (O13681) and Ima1/Samp1/Net5 have been determined (Varberg et al. 2020).

The favored 'selective phase model' (Hülsmann et al., 2012) assumes that the barrier-forming FG domains comprise many cohesive units, which bind each other and thereby mediate multivalent interactions within and between individual FG domains. Such interactions result in a sieve-like FG hydrogel that allows passage of small molecules but suppresses fluxes of larger ones. NTRs overcome this size limit by binding to FG motifs and consequently disengaging FG meshes in their immediate vicinity. This way, NTRs can partition into the FG hydrogel and exit the barrier on the trans side (Hülsmann et al., 2012).

Nuclear export of mRNAs was thought to occur exclusively through nuclear pore complexes. However, Speese et al. (2012) identified an alternate pathway for mRNA export in muscle cells where ribonucleoprotein complexes involved in forming neuromuscular junctions transit the nuclear envelope by fusing with and budding through the nuclear membrane.

Rothballer and Kutay (2013) have discussed the biogenesis of NPCs during interphase of the cell cycle. This process requires a mechanistically enigmatic fusion step between the inner and the outer nuclear membrane. They focus on the principle of membrane pore formation in the nuclear envelope and consider existing paradigms of other cellular membrane remodeling events. The emerging roles of transmembrane proteins and membrane-shaping factors in NPC biogenesis are considered. Transport of integral proteins to the innwe nuclear membrane involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex (Ohba et al. 2004).

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements (Rothballer et al. 2013). LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have revealed the molecular basis of nucleo-cytoskeletal coupling (Rothballer et al., 2013). They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. 

The majority of nuclear import pathways are mediated by importin-cargo interactions, but not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. Lu et al. 2014 identify a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy, leading to nuclear accumulation. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway used by AR-containing transcriptional regulators (Lu et al. 2014). 

Stuwe et al. 2015 presented the reconstitution of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. They proposed that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation (Stuwe et al. 2015). 

NPCs mediate nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Chug et al. 2015 reported a structural analysis of the frog FG Nup62•58•54 complex. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. Chug et al. 2015 further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. Chug et al. 2015 suggested that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Ciliates have two functionally distinct nuclei, a transcriptionally active somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Iwamoto et al. 2015 showed, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Addition of new nuclear envelope (NE) precursors occured through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD resulted in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Thus, NPC type switching plays a crucial role in the establishment of nuclear differentiation in ciliates (Iwamoto et al. 2015). 

The NPCs in ciliates consists of about 30 different nucleoporins each. Iwamoto et al. 2017 presented evidence for compositionally distinct NPCs that form within a single cell of Tetrahymena thermophila. Each cell contains both a MAC and a MIC. Iwamoto et al. 2017 identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs, and the transmembrane components, Pom121 and Pom82, localize exclusively to MAC and MIC NPCs, respectively. Thus, functional nuclear dimorphism in ciliates depends on compositional and structural differences. Neurocystin (NPHP) proteins (e.g., O15259) anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle (Blasius et al. 2019).

A protein known to localize to and be important in the assembly of both the yeast NPC and the spindle pole body, which functions as the microtubule organizing center, is the 6 TMS protein, Ndc1p (NPC1 in humans). The N- and C-termini of Ndc1p are exposed to the cytoplasm (Lau et al. 2006). The paralogous Brr6 and Brl1 are conserved integral membrane proteins of the nuclear envelope (NE). Depletion of Brr6 and Brl1 caused defects in NPC biogenesis, whereas the already assembled NPCs remained unaffected. This NPC biogenesis defect was not accompanied by a change in lipid composition. However, Brl1 interacted with Ndc1 and Nup188 as well as transmembrane and outer and inner ring NPC components, indicating a direct role in NPC biogenesis.Both Brr6 and Brl1 associated with a subpopulation of NPCs and emerging NPC assembly sites. BRL1 overexpression affected NE morphology and suppressed the nuclear pore biogenesis defect of Δnup116 and Δgle2 cells. Possibly Brr6 and Brl1 transiently associate with NPC assembly sites where they promote NPC biogenesis (Zhang et al. 2018).

Importin-beta, Ran GTPase (Ran), RanGAP, and RanBP2 have been identified as proteins interaction with a thioredoxin-like protein, TMX2 (Oguro and Imaoka 2019). Importin-beta is an adaptor protein which imports cargoes from the cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-beta complex, which allows the recycling of importin-beta and reentry of Ran into the nucleus. Oguro and Imaoka 2019 showed that TMX2 is not a transport cargo. It localizes in the outer nuclear membrane with its N- and C-termini facing the cytoplasm, where it co-localizes with importin-beta and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and cysteine 112 of Ran is important in its regulation by TMX2. Knockdown of TMX2 suppressed importin-beta-mediated transport of proteins. Thus, TMX2 works as a regulator of protein nuclear transport, and it facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins (Oguro and Imaoka 2019).

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane. Mudumbi et al. 2020 distinguished protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains, containing nuclear localization signals, into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels without blocking their translocation using the peripheral channels. Blocking the peripheral channels blocks translocation through both channels (Mudumbi et al. 2020). In the June 10th 2022 issue of Science, a written symposium appeared focusing on structural aspects of the NPC. Emphasis was placed on the Cytoplasmic Ring. Several additional proteins were identified (Bley et al. 2022; Petrovic et al. 2022).

The bi-directional nucleocytoplasmic shuttling of macromolecules like molecular signals, transcription factors, regulatory proteins, and RNAs occurs exclusively through Nuclear Pore Complexes (NPC) residing in the nuclear membrane. This complex is essentially a congregation of ~32 conserved proteins termed Nucleoporins (Nups) present in multiple copies and mostly arranged as subcomplexes to constitute a functional NPC (Bindra and Mishra 2021). Nups participate in ancillary functions such as chromatin organization, transcriptional regulation, DNA damage repair, genome stabilization, and cell cycle control, in addition to their central role as nucleocytoplasmic conduits. Thus, Nups play a role in the maintenance of cellular homeostasis. In mammals, three nucleoporins that traverse the nuclear membrane are called transmembrane Nups (TM-Nups) and are involved in multiple cellular functions. They are NUP35 (also called MP44) of 326 aas with 1 or 2 possible central TMSs, NUP219 of 1887 aas and 2 TMSs, N- and C-terminal, and NUP121 (POM121) of 1249 aas with two N-terminal TMSs. Owing to their vital roles in cellular processes and homeostasis, dysregulation of nucleoporin function is implicated in various diseases such as cancer. Bindra and Mishra 2021 emphasize the distinct canonical and non-canonical functions of mammalian TM-Nups and the underlying mechanisms of their disease association.

The NPC is structurally conserved across eukaryotes as are many of the pore's constituent proteins. The transmembrane nuclear pore proteins, GP210 and NDC1, span the nuclear envelope holding,,the nuclear pore in place. Orthologues of GP210 and NDC1 in Arabidopsis were investigated through characterisation of T-DNA insertional mutants. While the T-DNA insert into GP210 reduced expression of the gene, the insert in the NDC1 gene resulted in increased expression in both the ndc1 mutant as well as the ndc1/gp210 double mutant (Collins et al. 2023). The ndc1 and gp210 individual mutants showed little phenotypic difference from wild-type plants, but the ndc1/gp210 mutant showed a range of phenotypic effects. As with many plant nuclear pore protein mutants, these effects included non-nuclear phenotypes such as reduced pollen viability, reduced growth and glabrous leaves in mature plants. The ndc1/gp210 double mutant exhibited nuclear-specific effects including modifications to nuclear shape in different cell types. Functional changes to nuclear transport in ndc1/gp210 plants were observed. The lack of phenotypes in individual insertional lines, and the relatively mild phenotype suggest that additional transmembrane nucleoporins, such as the recently-discovered CPR5, likely compensate for their loss (2012) identified an alternate pathway for mRNA export in muscle cells where ribonucleoprotein complexes involved in forming neuromuscular junctions transit the nuclear envelope by fusing with and budding through the nuclear membrane.

Rothballer and Kutay (2013) have discussed the biogenesis of NPCs during interphase of the cell cycle. This process requires a mechanistically enigmatic fusion step between the inner and the outer nuclear membrane. They focus on the principle of membrane pore formation in the nuclear envelope and consider existing paradigms of other cellular membrane remodeling events. The emerging roles of transmembrane proteins and membrane-shaping factors in NPC biogenesis are considered. Transport of integral proteins to the innwe nuclear membrane involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex (Ohba et al. 2004).

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements (Rothballer et al. 2013). LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have revealed the molecular basis of nucleo-cytoskeletal coupling (Rothballer et al., 2013). They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. 

The majority of nuclear import pathways are mediated by importin-cargo interactions, but not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. Lu et al. 2014 identify a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy, leading to nuclear accumulation. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway used by AR-containing transcriptional regulators (Lu et al. 2014). 

Stuwe et al. 2015 presented the reconstitution of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. They proposed that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation (Stuwe et al. 2015). 

NPCs mediate nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Chug et al. 2015 reported a structural analysis of the frog FG Nup62•58•54 complex. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. Chug et al. 2015 further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. Chug et al. 2015 suggested that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Ciliates have two functionally distinct nuclei, a transcriptionally active somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Iwamoto et al. 2015 showed, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Addition of new nuclear envelope (NE) precursors occured through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD resulted in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Thus, NPC type switching plays a crucial role in the establishment of nuclear differentiation in ciliates (Iwamoto et al. 2015). 

The NPCs in ciliates consists of about 30 different nucleoporins each. Iwamoto et al. 2017 presented evidence for compositionally distinct NPCs that form within a single cell of Tetrahymena thermophila. Each cell contains both a MAC and a MIC. Iwamoto et al. 2017 identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs, and the transmembrane components, Pom121 and Pom82, localize exclusively to MAC and MIC NPCs, respectively. Thus, functional nuclear dimorphism in ciliates depends on compositional and structural differences. Neurocystin (NPHP) proteins (e.g., O15259) anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle (Blasius et al. 2019).

A protein known to localize to and be important in the assembly of both the yeast NPC and the spindle pole body, which functions as the microtubule organizing center, is the 6 TMS protein, Ndc1p (NPC1 in humans). The N- and C-termini of Ndc1p are exposed to the cytoplasm (Lau et al. 2006). The paralogous Brr6 and Brl1 are conserved integral membrane proteins of the nuclear envelope (NE). Depletion of Brr6 and Brl1 caused defects in NPC biogenesis, whereas the already assembled NPCs remained unaffected. This NPC biogenesis defect was not accompanied by a change in lipid composition. However, Brl1 interacted with Ndc1 and Nup188 as well as transmembrane and outer and inner ring NPC components, indicating a direct role in NPC biogenesis.Both Brr6 and Brl1 associated with a subpopulation of NPCs and emerging NPC assembly sites. BRL1 overexpression affected NE morphology and suppressed the nuclear pore biogenesis defect of Δnup116 and Δgle2 cells. Possibly Brr6 and Brl1 transiently associate with NPC assembly sites where they promote NPC biogenesis (Zhang et al. 2018).

Importin-beta, Ran GTPase (Ran), RanGAP, and RanBP2 have been identified as proteins interaction with a thioredoxin-like protein, TMX2 (Oguro and Imaoka 2019). Importin-beta is an adaptor protein which imports cargoes from the cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-beta complex, which allows the recycling of importin-beta and reentry of Ran into the nucleus. Oguro and Imaoka 2019 showed that TMX2 is not a transport cargo. It localizes in the outer nuclear membrane with its N- and C-termini facing the cytoplasm, where it co-localizes with importin-beta and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and cysteine 112 of Ran is important in its regulation by TMX2. Knockdown of TMX2 suppressed importin-beta-mediated transport of proteins. Thus, TMX2 works as a regulator of protein nuclear transport, and it facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins (Oguro and Imaoka 2019).

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane. Mudumbi et al. 2020 distinguished protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains, containing nuclear localization signals, into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels without blocking their translocation using the peripheral channels. Blocking the peripheral channels blocks translocation through both channels (Mudumbi et al. 2020). In the June 10th 2022 issue of Science, a written symposium appeared focusing on structural aspects of the NPC. Emphasis was placed on the Cytoplasmic Ring. Several additional proteins were identified (Bley et al. 2022; Petrovic et al. 2022).

The bi-directional nucleocytoplasmic shuttling of macromolecules like molecular signals, transcription factors, regulatory proteins, and RNAs occurs exclusively through Nuclear Pore Complexes (NPC) residing in the nuclear membrane. This complex is essentially a congregation of ~32 conserved proteins termed Nucleoporins (Nups) present in multiple copies and mostly arranged as subcomplexes to constitute a functional NPC (Bindra and Mishra 2021). Nups participate in ancillary functions such as chromatin organization, transcriptional regulation, DNA damage repair, genome stabilization, and cell cycle control, in addition to their central role as nucleocytoplasmic conduits. Thus, Nups play a role in the maintenance of cellular homeostasis. In mammals, three nucleoporins that traverse the nuclear membrane are called transmembrane Nups (TM-Nups) and are involved in multiple cellular functions. They are NUP35 (also called MP44) of 326 aas with 1 or 2 possible central TMSs, NUP219 of 1887 aas and 2 TMSs, N- and C-terminal, and NUP121 (POM121) of 1249 aas with two N-terminal TMSs. Owing to their vital roles in cellular processes and homeostasis, dysregulation of nucleoporin function is implicated in various diseases such as cancer. Bindra and Mishra 2021 emphasize the distinct canonical and non-canonical functions of mammalian TM-Nups and the underlying mechanisms of their disease association.

The NPC is structurally conserved across eukaryotes as are many of the pore's constituent proteins. The transmembrane nuclear pore proteins, GP210 and NDC1, span the nuclear envelope holding,,the nuclear pore in place. Orthologues of GP210 and NDC1 in Arabidopsis were investigated through characterisation of T-DNA insertional mutants. While the T-DNA insert into GP210 reduced expression of the gene, the insert in the NDC1 gene resulted in increased expression in both the ndc1 mutant as well as the ndc1/gp210 double mutant (Collins et al. 2023). The ndc1 and gp210 individual mutants showed little phenotypic difference from wild-type plants, but the ndc1/gp210 mutant showed a range of phenotypic effects. As with many plant nuclear pore protein mutants, these effects included non-nuclear phenotypes such as reduced pollen viability, reduced growth and glabrous leaves in mature plants. The ndc1/gp210 double mutant exhibited nuclear-specific effects including modifications to nuclear shape in different cell types. Functional changes to nuclear transport in ndc1/gp210 plants were observed. The lack of phenotypes in individual insertional lines, and the relatively mild phenotype suggest that additional transmembrane nucleoporins, such as the recently-discovered CPR5, likely compensate for their loss (Collins et al. 2023).