Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

Interaction of nucleoporins with nuclear transport receptors: a structural perspective.

Soluble nuclear transport receptors and stationary nucleoporins are at the heart of the nucleocytoplasmic transport machinery. A subset of nucleoporins contains characteristic and repetitive FG (phenylalanine-glycine) motifs, which are the basis for the permeability barrier of the nuclear pore complex (NPC) that controls transport of macromolecules between the nucleus and the cytoplasm. FG-motifs can interact with each other and/or with transport receptors, mediating their translocation across the NPC. The molecular details of homotypic and heterotypic FG-interactions have been analyzed at the structural level. In this review, we focus on the interactions of nucleoporins with nuclear transport receptors. Besides the conventional FG-motifs as interaction spots, a thorough structural analysis led us to identify additional similar motifs at the binding interface between nucleoporins and transport receptors. A detailed analysis of all known human nucleoporins revealed a large number of such phenylalanine-containing motifs that are not buried in the predicted 3D-structure of the respective protein but constitute part of the solvent-accessible surface area. Only nucleoporins that are rich in conventional FG-repeats are also enriched for these motifs. This additional layer of potential low-affinity binding sites on nucleoporins for transport receptors may have a strong impact on the interaction of transport complexes with the nuclear pore and, thus, the efficiency of nucleocytoplasmic transport.

Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein.

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/ß. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-ß, -7, -ß/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.

RAPIDS, a method for sub-compartmental identification of protein interactomes.

Protein-protein interactions are central to most cellular processes and their dysregulation has been related to the development of various diseases. Proximity-based labeling methods are used to identify the endogenous interaction partners of specific proteins of interest (POIs). The POI is fused to promiscuous enzymes, which generate reactive species in vivo and label proteins in close vicinity. APEX-based proximity labeling techniques utilize an engineered ascorbate peroxidase, which in the presence of H2O2 oxidizes biotin-phenol to short lived biotin-phenoxyl radicals that biotinylate nearby proteins. The biotinylated proteins are enriched by biotin affinity capture and identified by mass spectrometry. We devised an advanced method, RAPIDS, in which the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together. RAPIDS improves the specificity of APEX-based interactome analysis by strictly eliminating false positives. In this chapter, we describe this method in detail, with VAPB as a protein of interest and versions of APEX2 with different subcellular localizations. VAPB localizing to different cellular compartments, the endoplasmic reticulum and the inner nuclear membrane, yielded distinct sets of proximity partners as identified by RAPIDS.

CRM1 Promotes Capsid Disassembly and Nuclear Envelope Translocation of Adenovirus Independently of Its Export Function.

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.

Sequestosome 1 Is Part of the Interaction Network of VAPB.

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments.

The Interactome of the VAP Family of Proteins: An Overview.

Membrane contact sites (MCS) are sites of close apposition of two organelles that help in lipid transport and synthesis, calcium homeostasis and several other biological processes. The VAMP-associated proteins (VAPs) VAPA, VAPB, MOSPD2 and the recently described MOSPD1 and MOSPD3 are tether proteins of MCSs that are mainly found at the endoplasmic reticulum (ER). VAPs interact with various proteins with a motif called FFAT (two phenylalanines in an acidic tract), recruiting the associated organelle to the ER. In addition to the conventional FFAT motif, the recently described FFNT (two phenylalanines in a neutral tract) and phospho-FFAT motifs contribute to the interaction with VAPs. In this review, we summarize and compare the recent interactome studies described for VAPs, including in silico and proximity labeling methods. Collectively, the interaction repertoire of VAPs is very diverse and highlights the complexity of interactions mediated by the different FFAT motifs to the VAPs.

The RNA-binding protein FUS is chaperoned and imported into the nucleus by a network of import receptors.

Fused in sarcoma (FUS) is a predominantly nuclear RNA-binding protein with key functions in RNA processing and DNA damage repair. Defects in nuclear import of FUS have been linked to severe neurodegenerative diseases; hence, it is of great interest to understand this process and how it is dysregulated in disease. Transportin-1 (TNPO1) and the closely related transportin-2 have been identified as major nuclear import receptors of FUS. They bind to the C-terminal nuclear localization signal of FUS and mediate the protein's nuclear import and at the same time also suppress aberrant phase transitions of FUS in the cytoplasm. Whether FUS can utilize other nuclear transport receptors for the purpose of import and chaperoning has not been examined so far. Here, we show that FUS directly binds to different import receptors in vitro. FUS formed stable complexes not only with TNPO1 but also with transportin-3, importin ß, importin 7, or the importin ß/7 heterodimer. Binding of these alternative import receptors required arginine residues within FUS-RG/RGG motifs and was weakened by arginine methylation. Interaction with these importins suppressed FUS phase separation and reduced its sequestration into stress granules. In a permeabilized cell system, we further showed that transportin-3 had the capacity to import FUS into the nucleus, albeit with lower efficiency than TNPO1. Our data suggest that aggregation-prone RNA-binding proteins such as FUS may utilize a network of importins for chaperoning and import, similar to histones and ribosomal proteins.

A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex.

Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

Dual-Color Metal-Induced Energy Transfer (MIET) Imaging for Three-Dimensional Reconstruction of Nuclear Envelope Architecture.

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and control transport of macromolecules between the two compartments. Recently, it has been shown that the axial distance between the inner nuclear membrane and the cytoplasmic side of the NPC can be measured using dual-color metal-induced energy transfer (MIET). This chapter focuses on experimental aspects of this method and discusses the details of data analysis.

The SQSTM1-NUP214 fusion protein interacts with Crm1, activates Hoxa and Meis1 genes, and drives leukemogenesis in mice.

The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the phenylalanine-glycine (FG) repeat motifs of the NUP moiety that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we took advantage of the Sequestosome-1 (SQSTM1)-NUP214 fusion. SQSTM1-NUP214 retains only a short C-terminal portion of NUP214 which contains FG motifs that mediate interaction with Crm1. We introduced point mutations targeting these FG motifs and found that the ability of the resulting SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 impaired myeloid maturation and conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was considerably diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice, whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia. These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impaired SQSTM1-NUP214 interaction with Crm1 correlated with impaired binding of the fusion protein to Hoxa and Meis1 genes. These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling.

Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.

Nup358 and Transportin 1 Cooperate in Adenoviral Genome Import.

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA.IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

Into the basket and beyond: the journey of mRNA through the nuclear pore complex.

The genetic information encoded in nuclear mRNA destined to reach the cytoplasm requires the interaction of the mRNA molecule with the nuclear pore complex (NPC) for the process of mRNA export. Numerous proteins have important roles in the transport of mRNA out of the nucleus. The NPC embedded in the nuclear envelope is the port of exit for mRNA and is composed of ∼30 unique proteins, nucleoporins, forming the distinct structures of the nuclear basket, the pore channel and cytoplasmic filaments. Together, they serve as a rather stationary complex engaged in mRNA export, while a variety of soluble protein factors dynamically assemble on the mRNA and mediate the interactions of the mRNA with the NPC. mRNA export factors are recruited to and dissociate from the mRNA at the site of transcription on the gene, during the journey through the nucleoplasm and at the nuclear pore at the final stages of export. In this review, we present the current knowledge derived from biochemical, molecular, structural and imaging studies, to develop a high-resolution picture of the many events that culminate in the successful passage of the mRNA out of the nucleus.

Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane.

Vesicle-associated membrane protein-associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.

The nuclear pore proteins Nup88/214 and T-cell acute lymphatic leukemia-associated NUP214 fusion proteins regulate Notch signaling.

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.

Targeting of LRRC59 to the Endoplasmic Reticulum and the Inner Nuclear Membrane.

LRRC59 (leucine-rich repeat-containing protein 59) is a tail-anchored protein with a single transmembrane domain close to its C-terminal end that localizes to the endoplasmic reticulum (ER) and the nuclear envelope. Here, we investigate the mechanisms of membrane integration of LRRC59 and its targeting to the inner nuclear membrane (INM). Using purified microsomes, we show that LRRC59 can be post-translationally inserted into ER-derived membranes. The TRC-pathway, a major route for post-translational membrane insertion, is not required for LRRC59. Like emerin, another tail-anchored protein, LRRC59 reaches the INM, as demonstrated by rapamycin-dependent dimerization assays. Using different approaches to inhibit importin α/ß-dependent nuclear import of soluble proteins, we show that the classic nuclear transport machinery does not play a major role in INM-targeting of LRRC59. Instead, the size of the cytoplasmic domain of LRRC59 is an important feature, suggesting that targeting is governed by passive diffusion.

The cargo spectrum of nuclear transport receptors.

The molecular mechanisms of nuclear transport have been described in great detail and we are beginning to understand the structures of transport complexes and even of subcomplexes of the nuclear pore at an atomic or near-atomic resolution. The complexity of the clients that use the transport machinery, by contrast, is less well understood, although some transport receptors are reported to have hundreds of different cargoes and others only a few. Here, we review the recent attempts to define the cargo spectrum of individual nuclear transport receptors using bioinformatic, biochemical and cell biological approaches and compare the results obtained by these complementary methods. Remarkably, a large fraction of the soluble proteome can be subject to nucleocytoplasmic transport.

Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence.

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

GCN5L1 interacts with αTAT1 and RanBP2 to regulate hepatic α-tubulin acetylation and lysosome trafficking.

Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.