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Abstract The nuclear pore membrane protein 121 (POM121) was originally thought to be a constitutive protein of the nuclear pore complex (NPC). In addition to being involved in NPC assembly, abnormal POM121 expression has been found to be associated with many diseases. In this study, we explored, in detail, the effect of POM121 on the macrophage inflammatory response and found that its expression was significantly lower in LPS-stimulated macrophages, substantially amplifying pro-inflammatory cytokine (TNF-α and IL-6) production, suggesting that POM121 exerts a potent inhibitory effect on macrophage inflammation. Consistent with this notion, greater susceptibility to LPS-induced acute lung injury (ALI) as well as more severe tissue inflammation were found in POM121fl/fl Lyzm-Cre+ mice compared to those in control mice, as evidenced by the more severe lung injury and inflammation, increased TNF-α and IL-6 production and more abundant proteins in bronchoalveolar lavage fluid (BALF). This inflammation-modulating effect of POM121 relied on its ability to repress the NF-κB signal pathway via inhibition of phosphorylated P65 (phos-P65) nuclear accumulation. In the present study, we reported that in addition to acting as a constitutive NPC component, POM121 also modulated LPS-induced macrophage inflammation via repressing nuclear P65 translocation. Our study may pave the way for regulating LPS-induced massive macrophage inflammation and providing evidence for the functional diversity of nucleoporins (Nups). Highlights • POM121 is down-regulated in LPS-stimulated macrophages. • POM121 inhibits the macrophage inflammatory response. • Macrophage POM121 deficient mice show more susceptibility to LPS-inducted acute lung injury. • POM121 selectively inhibits nuclear accumulation of NF-κB p65. [ABSTRACT FROM AUTHOR]

Nucleoporins are a specialized subset of nuclear proteins that comprise the nuclear pore complex and regulate nucleocytoplasmic transport. Recent demonstrations of roles for individual nucleoporins in multiple paradigms of differentiation via mechanisms independent of nuclear trafficking represent conceptual advances in understanding the contributions of nucleoporins to cellular development. Among these, a functional role for nucleoporins in reproductive fitness and gametogenesis has been identified, supported by robust models and clinical studies that leverage the power of next generation sequencing technology to identify reproductive-disease-associated mutations in specific nucleoporins. Proper nucleoporin function manifests in different ways during oogenesis and spermatogenesis. However, nonhuman models of gametogenesis may not recapitulate human mechanisms, which may confound translational interpretation and relevance. To circumvent these limitations, identification of reproductive pathologies in patients, combined with next generation sequencing approaches and advanced in silico tools, offers a powerful approach to investigate the potential function of nucleoporins in human reproduction. Ultimately, elucidating the role of nucleoporins in reproductive biology will provide opportunities for predictive, diagnostic, and therapeutic strategies to address reproductive disorders. [ABSTRACT FROM AUTHOR]

Highlights • The nuclear envelope resists quantitative study by conventional light microscopy. • Super-resolution images contain sufficient data to allow precise numerical analysis. • An algorithm to quantify nuclear pore complex and lamina distribution is described. • The algorithm can be easily adapted to the analysis of other cellular components. Abstract For decades, components of the mammalian nuclear envelope (NE), such as the nuclear lamina and nuclear pore complexes (NPCs), have been largely resistant to quantitative cell biological analysis using conventional fluorescence microscopy. This is in part due to their sub diffraction limit dimensions. Super-resolution microscopy, a major advancement in cell biology research, has now made possible the acquisition of images in which nuclear lamin networks and single NPCs can be resolved in intact mammalian somatic cells. In particular, single molecule localization microscopy is able to generate data sets that are accurate enough to allow detailed quantitative analysis. Here we describe an algorithm that will identify the centroid of single NPCs and will determine their localization relative to the distribution of lamin protein filaments. Using this algorithm, a percentage of NPCs localized within the nuclear lamin network was accurately calculated, that could be compared between cells expressing different lamin complements. With modifications tweaked according to user specified sample images, this algorithm serves as a semi-automatic and fast computational tool to quantify and compare the localization and distribution of two or more cellular components at the nanometre scale. [ABSTRACT FROM AUTHOR]

Nuclear pore complex (NPC) is a fundamental component of the nuclear envelope and is key to the nucleocytoplasmic transport. Mutations in several NUP genes that encode individual components of NPC known as nucleoporins have been identified in recent years among patients with static encephalopathies characterized by developmental delay and microcephaly. We describe a multiplex consanguineous family in which four affected members presented with severe neonatal hypotonia, profound global developmental delay, progressive microcephaly and early death. Autozygome and linkage analysis revealed that this phenotype is linked to a founder disease haplotype (chr9:127,113,732-135,288,807) in which whole exome sequencing revealed the presence of a novel homozygous missense variant in NUP214. Functional analysis of patient-derived fibroblasts recapitulated the dysmorphic phenotype of nuclei that was previously described in NUP214 knockdown cells. In addition, the typical rim staining of NUP214 is largely displaced, further supporting the deleterious effect of the variant. Our data expand the list of NUP genes that are mutated in encephalopathy disorders in humans. [ABSTRACT FROM AUTHOR]

Nuclear pore complex (NPC) is the largest multimeric protein assembly of the eukaryotic cell, which mediates the nucleocytoplasmic transport. The constituent proteins of this assembly (nucleoporins) are present in varying copy numbers to give a size from ~ 60 MDa (yeast) to 112 MDa (human) and share common ancestry with other membrane‐associated complexes such as COPI/COPII and thus share the same structural folds. However, the nucleoporins across species exhibit very low percentage sequence similarity and this reflects in their distinct secondary structure and domain organization. We employed thorough sequence and phylogenetic analysis guided from structure‐based alignments of all the nucleoporins from fungi to metazoans to understand the evolution of NPC. Through evolutionary pressure analysis on various nucleoporins, we deduced that these proteins are under differential selection pressure and hence the homologous interacting partners do not complement each other in the in vitro pull‐down assay. The super tree analysis of all nucleoporins taken together illustrates divergent evolution of nucleoporins and notably, the degree of divergence is more apparent in higher order organisms as compared to lower species. Overall, our results support the hypothesis that the protein–protein interactions in such large multimeric assemblies are species specific in nature and hence their structure and function should also be studied in an organism‐specific manner. [ABSTRACT FROM AUTHOR]

The partitioning of the proteome between nucleus and cytoplasm affects nearly every aspect of eukaryotic biology. Despite this central role, we still have a poor understanding of which proteins localize in the nucleus and how this varies in different cell types and conditions. Recent advances in quantitative proteomics and high-throughput imaging are starting to close this knowledge gap. Studies on protein interaction are beginning to reveal the spectrum of cargos of nuclear import and export receptors. We anticipate that it will soon be possible to predict each protein's nucleocytoplasmic localization based on its importin/exportin interactions and its estimated diffusion rate through the nuclear pore. This insight is likely to provide us with a fundamental understanding of how cells use nucleocytoplasmic partitioning to encode and relay information. [ABSTRACT FROM AUTHOR]

Graphical abstract Nuclear export of individual mRNAs through nuclear pore complexes revealed by SPEED microscopy. Abstract The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs. In this review, we firstly evaluate the necessity of single-molecule live-cell microscopy in the study of mRNA nuclear export. Then, we highlight the application of single-point edge-excitation sub-diffraction (SPEED) microscopy that combines high-speed SMF microscopy and a 2D-to-3D transformation algorithm in the studies of nuclear transport kinetics and route for mRNAs. Finally, we summarize the new features of mRNA nuclear export found with SPEED microscopy as well as the reliability and accuracy of SPEED microscopy in mapping the 3D spatial locations of transport routes adopted by proteins and mRNAs through the NPCs. [ABSTRACT FROM AUTHOR]

Key message: Loss-of-function of nucleoporin NUP1 in Arabidopsis causes defect in both male and female gametogenesis. Its ovules are arrested during meiosis, and its pollen grains are aborted at mitosis I.Abstract: Nuclear pore complex (NPC) plays crucial roles in nucleocytoplasmic trafficking of proteins and RNAs. The NPC contains approximately 30 different proteins termed nucleoporins (NUPs). So far, only a few of plant NUPs have been characterized. The Arabidopsis NUP1 was identified as an ortholog of the yeast NUP1 and animal NUP153. Loss-of-function of NUP1 in Arabidopsis caused fertility defect; however, the molecular mechanism of this defect remains unknown. Here, we found that both male and female gametogenesis of the nup1 mutants were defective. nup1 ovules were arrested from the meiosis stage onward; only approximately 6.7% and 3% ovules of the nup1-1 and nup1-4 mutants developed up to the FG7 stage, respectively. Pollen development of the nup1 mutants was arrested during the first mitotic division. In addition, enlarged pollen grains with increased DNA content were observed in the nup1 mutant. RNA-sequencing showed that expression levels of genes involved in pollen development or regulation of cell size were reduced dramatically in nup1 compared with wild type. These results suggest that NUP1 plays an important role in gametogenesis. [ABSTRACT FROM AUTHOR]

In biological cells, nuclear pore complexes (NPCs) embedded in cell membranes are capable of controlling the flow of ions, for example, Na+, K+, and Ca2+ by responding to stimuli, for example, pH and voltage. Inspired by NPCs, researchers have been endeavoring to develop nanogates to achieve the control of ion transport, but the developed nanogates only have a low factor of change in ion currents due to ON/OFF switching. As such nanopores with high temperature and pH responsivities were developed in this work. According to the experimental results, at a voltage of 3 V, the change in ion currents due to pH change is up to a factor of 170, which is remarkably high compared to other nanogates reported. Quantum chemical (QC) calculation results show that a protonated cytosine molecule (C+) and an unprotonated cytosine molecule (C) form three pairs of hydrogen bonds and consequently a nucleobase pair, CC+, leading to the binding of various strands, assembly of a strand net, and blockage of ion transport. The nanogate developed is capable of responding to temperature change. At a voltage of 3 V, the factor of change in ion currents in response to temperature variation is as high as 110. Further experiments were performed to investigate the influence of the NaCl concentrations and small opening diameters exerted on nanogate performance. Inspired by nuclear pore complexes (NPCs), researchers have been endeavoring to develop nanogates to achieve the control of ion transport, but developed nanogates only have a low factor of change in ion currents due to ON/OFF switching. Nanopores with high temperature and pH responsivities were developed in the work. [ABSTRACT FROM AUTHOR]

The nuclear pore complex is the main transportation hub for exchange between the cytoplasm and the nucleus. It is built from nucleoporins that form distinct subcomplexes to establish this huge protein complex in the nuclear envelope. Malfunctioning of nucleoporins is well known in human malignancies, such as gene fusions of NUP214 and NUP98 in hematological neoplasms and overexpression of NUP88 in a variety of human cancers. In the past decade, the incremental utilization of next-generation sequencing has unraveled mutations in nucleoporin genes in the context of an increasing number of hereditary diseases, often in a tissue-specific manner. It emerges that, on one hand, the central nervous system and the heart are particularly sensitive to mutations in nucleoporin genes. On the other hand, nucleoporins forming the scaffold structure of the nuclear pore complex are eminently mutation-prone. These novel and exciting associations between nucleoporins and human diseases emphasize the need to shed light on these unanticipated tissue-specific roles of nucleoporins that may go well beyond their role in nucleocytoplasmic transport. In this review, the current insights into altered nucleoporin function associated with human hereditary disorders will be discussed. [ABSTRACT FROM AUTHOR]

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution. [Display omitted] • A composite multiscale 3D structure of the isolated yeast NPC has been determined • Double nuclear outer rings represent an ancient feature of NPCs • Membrane anchor sites on the inner ring use a nucleoporin β-propeller/TMD partner motif • Radial expansion of the NPC scaffold unplugs the central channel to promote transport Akey et al. report a multiscale structure of the isolated yeast NPC that provides a framework to piece together the roles of nucleoporins in assembly and transport while providing insights into the evolution of critical sub-assemblies. [ABSTRACT FROM AUTHOR]

It has been shown that the nucleoporin ALADIN plays a significant role in the redox homeostasis of the cell, but its function in steroidogenesis contributing to adrenal atrophy in triple A syndrome remains largely unknown. In an attempt to identify new interaction partners of ALADIN, co-immunoprecipitation followed by proteome analysis was conducted in different expression models using the human adrenocortical tumour cell line NCI-H295R. Our results suggest an interaction of ALADIN with the microsomal protein PGRMC2. PGRMC2 is shown to be activity regulator of CYP P450 enzymes and, therefore, to be a possible target for adrenal dysregulation in triple A syndrome. We show that there is a sexual dimorphism regarding the expression of Pgrmc2 in adrenals and gonads of wild-type (WT) and Aaas knock-out (KO) mice. Female Aaas KO mice are sterile due to delayed oocyte maturation and meiotic spindle assembly. A participation in meiotic spindle assembly confirms the recently investigated involvement of ALADIN in mitosis and emphasises an interaction with PGRMC2 which is a regulator of the cell cycle. By identification of a novel interaction partner of ALADIN, we provide novel aspects for future research of the function of ALADIN during cell cycle and for new insights into the pathogenesis of triple A syndrome.

FG nucleoporins (Nups) are the class of proteins that both generate the permeability barrier and mediate selective transport through the nuclear pore complex (NPC). The FG Nup family has 11 members in Saccharomyces cerevisiae, and the study of mutants lacking different FG domains has been instrumental in testing transport models. To continue analyzing the distinct functional roles of FG Nups in vivo, additional robust genetic tools are required. Here, we describe a novel collection of S. cerevisiae mutant strains in which the FG domains of different groups of Nups are absent (Δ) in the greatest number documented to date. Using this plasmid-based ΔFG strategy, we find that a GLFG domain-only pore is sufficient for viability. The resulting extensive plasmid and strain resources are available to the scientific community for future in-depth in vivo studies of NPC transport.

Our view of the nuclear pore complexes (NPCs) as gateways between the nuclear and cytoplasmic compartments has been largely expanded in recent years. NPCs have now demonstrated roles in genome regulation and maintenance from single cells to multicellular organisms. Both NPC proteins as well as components of the NPC basket act as dynamic scaffolds for silencing factors, and chromatin and cell cycle regulators. Components of the NPC basket also couple mRNA production and export, and prevent the exit of unprocessed mRNAs from the nucleus. Our recent work describes a novel function of the fission yeast nuclear basket component – the translocated promoter region (TPR) nucleoporin Alm1 – in proper localization of the proteasome to the nuclear envelope. Here we discuss how regulation of proteasome localization to the nuclear envelope by Alm1 is key to maintain kinetochores homeostasis and proper chromosome segregation.

During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.

BioID is a well-established method for identifying protein–protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15–18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.

The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

A recent advance in understanding stem cell differentiation is that the cell is able to translate its morphology, i.e., roundish or spread, into a fate decision. We hypothesize that strain states in the nuclear envelope (NE) cause changes in the structure of the nuclear pore complexes. This induces significant changes in the NE's permeability to the traffic of the transcription factors involved in stem cell differentiation which are imported into the nucleus by passive diffusion. To demonstrate this, we set up a numerical model of the transport of diffusive molecules through the nuclear pore complex (NPC), on the basis of the NPC deformation. We then compared the prediction of the model for two different cell configurations with roundish and spread nuclear topologies with those measured on cells cultured in both configurations. To measure the geometrical features of the NPC, using electron tomography we reconstructed three-dimensional portions of the envelope of cells cultured in both configurations. We found non-significant differences in both the shape and size of the transmembrane ring of single pores with envelope deformation. In the numerical model, we thus assumed that the changes in pore complex permeability, caused by the envelope strains, are due to variations in the opening configuration of the nuclear basket, which in turn modifies the porosity of the pore complex mainly on its nuclear side. To validate the model, we cultured cells on a substrate shaped as a spatial micro-grid, called the “nichoid,” which is nanoengineered by two-photon laser polymerization, and induces a roundish nuclear configuration in cells adhering to the nichoid grid, and a spread configuration in cells adhering to the flat substrate surrounding the grid. We then measured the diffusion through the nuclear envelope of an inert green-fluorescent protein, by fluorescence recovery after photobleaching (FRAP). Finally, we compared the diffusion times predicted by the numerical model for roundish vs. spread cells, with the measured times. Our data show that cell stretching modulates the characteristic time needed for the nuclear import of a small inert molecule, GFP, and the model predicts a faster import of diffusive molecules in the spread compared to roundish cells.

Nuclear pore complexes (NPCs) lined with intrinsically disordered FG-domains act as selective gatekeepers for molecular transport between the nucleus and the cytoplasm in eukaryotic cells. The underlying physical mechanism of the intriguing selectivity is still under debate. Here, we probe the transport of ions and transport receptors through biomimetic NPCs consisting of Nsp1 domains attached to the inner surface of solid-state nanopores. We examine both wildtype FG-domains and hydrophilic SG-mutants. FG-nanopores showed a clear selectivity as transport receptors can translocate across the pore whereas other proteins cannot. SG mutant pores lack such selectivity. To unravel this striking difference, we present coarse-grained molecular dynamics simulations that reveal that FG-pores exhibit a high-density, nonuniform protein distribution, in contrast to a uniform and significantly less-dense protein distribution in the SG-mutant. We conclude that the sequence-dependent density distribution of disordered proteins inside the NPC plays a key role for its conductivity and selective permeability.

The nuclear pore complex (NPC) is the sole gateway for molecular transport between the nucleus and the cytoplasm in eukaryotes. The NPC is composed of approximately 30 different kinds of protein components called nucleoporins. The functional structure of the NPC is highly conserved among various eukaryotes. However, the exact mechanisms by which many of the nucleoporins exert their specific functions still remain unclear. The single cell ciliate Tetrahymena has two functionally distinct nuclei, a macronucleus and a micronucleus, and we have discovered that these two nuclei have compositionally distinct NPCs. We initially identified four nucleus-specific Nup98-s and demonstrated that they are required for macronucleus- and micronucleus-specific nuclear transport. More recently we identified two nucleus-specific paralogs of Nup214 and Nup153 and two nucleus-specific transmembrane proteins Pom121 and Pom82. Our findings argue that Nup214, Nup153, and Pom121/Pom82 also act as key molecules for transport machineries to differentiate the two functionally distinct nuclei present in this organism.

Oocytes stockpile nuclear pore complexes (NPCs) in cytoplasmic membrane sheets called annulate lamellae (AL) in preparation for rapid cell cycles during embryogenesis. Recently, Hampoelz et al. reported that AL–NPC assembly depends on the coordinated formation, transport, and interaction of biomolecular condensates containing distinct sets of nucleoporins. [ABSTRACT FROM AUTHOR]

The segregation of the chromosomes during mitosis is an important process, in which the replicated DNA content is properly allocated into two daughter cells. To ensure their genomic integrity, cells present an essential surveillance mechanism known as the spindle assembly checkpoint (SAC), which monitors the bipolar attachment of the mitotic spindle to chromosomes to prevent errors that would result in chromosome mis-segregation and aneuploidy. Multiple components of the nuclear pore complex (NPC), a gigantic protein complex that forms a channel through the nuclear envelope to allow nucleocytoplasmic exchange of macromolecules, were shown to be critical for faithful cell division and implicated in the regulation of different steps of the mitotic process, including kinetochore and spindle assembly as well as the SAC. In this review, we will describe current knowledge about the interconnection between the NPC and the SAC in an evolutional perspective, which primarily relies on the two mitotic checkpoint regulators, Mad1 and Mad2. We will further discuss the role of NPC constituents, the nucleoporins, in kinetochore and spindle assembly and the formation of the mitotic checkpoint complex during mitosis and interphase.

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast.

Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98–Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98–Nup96 joint molecule as a sole protein product of thenup189+ gene in the fission yeastSchizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98–Nup96 is dispensable for cell growth, at least under normal culture conditions inS. pombe.

Acute necrotizing encephalopathy 1 (ANE1) is a very rare disorder associated with a dominant heterozygous mutation in the RANBP2 (RAN binding protein 2) gene. ANE1 is frequently triggered by a febrile infection and characterized by serious and irreversible neurological damage. Although only a few hundred cases have been reported, mutations in RANBP2 are only partially penetrant and can occur de novo , suggesting that their frequency may be higher in some populations. Genetic diagnosis is a lengthy process, potentially delaying definitive diagnosis. We therefore developed a rapid bedside qPCR-based tool for early diagnosis and screening of ANE1 mutations. Primers were designed to specifically assess RANBP2 and not RGPD (RANBP2 and GCC2 protein domains) and discriminate between wild-type or mutant RANBP2 . Nasal epithelial cells were obtained from two individuals with known RANBP2 mutations and two healthy control individuals. RANBP2 -specific reverse transcription followed by allele-specific primer qPCR amplification confirmed the specific detection of heterozygously expressed mutant RANBP2 in the ANE1 samples. This study demonstrates that allele-specific qPCR can be used as a rapid and inexpensive diagnostic tool for ANE1 using preexisting equipment at local hospitals. It can also be used to screen non-hospitalized family members and at risk-population to better establish the frequency of non-ANE-associated RANBP2 mutations, as well as possible tissue-dependent expression patterns., Systematic Review Registration: The protocol was registered in the international prospective register of systematic reviews (PROSPERO- CRD42023443257)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gouy, Decorsière, Desgraupes, Duan, Ouyang, Wang, Yeh, Palazzo, Moraes, Nisole and Arhel.)