Browsing by Author "Spillner, Christiane"

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.

Abstract 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.

Nucleoporins containing phenylalanine glycine (FG) repeats play an important role in nucleocytoplasmic transport as they bind to transport receptors and mediate translocation of transport complexes across the nuclear pore complex (NPC). Nup214/CAN, a nucleoporin that is found at the cytoplasmic side of the NPC, interacts with both import and export receptors. In functional assays, dominant-negative fragments of Nup214 inhibited CRM1-dependent nuclear export, as the export receptor became rate-limiting. Several nuclear import pathways, by contrast, were not affected by the Nup214 fragments. We now characterize the CRM1-binding region of Nup214 in detail and identify several FG motives that are required for this interaction. Our results support a model where CRM1, like other transport receptors, contacts FG-Nups via multiple binding sites.

CRM1 is the major nuclear export receptor. During translocation through the nuclear pore, transport complexes transiently interact with phenylalanine-glycine (FG) repeats of multiple nucleoporins. On the cytoplasmic side of the nuclear pore, CRM1 tightly interacts with the nucleoporin Nup214. Here, we present the crystal structure of a 117-amino-acid FG-repeat-containing fragment of Nup214, in complex with CRM1, Snurportin 1, and RanGTP at 2.85 angstrom resolution. The structure reveals eight binding sites for Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor. Nup214 binds to N- and C-terminal regions of CRM1, thereby clamping CRM1 in a closed conformation and stabilizing the export complex. The role of conserved hydrophobic pockets for the recognition of FG motifs was analyzed in biochemical and cell-based assays. Comparative studies with RanBP3 and Nup62 shed light on specificities of CRM1-nucleoporin binding, which serves as a paradigm for transport receptor-nucleoporin interactions.

The signaling molecule 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) has been described as the "anti-inflammatory prostaglandin." Here we show that substrates of the nuclear export receptor CRM1 accumulate in the nucleus in the presence of 15d-PGJ(2), identifying this prostaglandin as a regulator of CRM1-dependent nuclear protein export that can be produced endogenously. Like leptomycin B (LMB), an established fungal CRM1-inhibitor, 15d-PGJ(2) reacts with a conserved cysteine residue in the CRM1 sequence. This covalent modification prevents the formation of nuclear export complexes. Cells that are transfected with mutant CRM1(C528S) are resistant to the inhibitory effects of LMB and 15d-PGJ(2), demonstrating that the same single amino acid is targeted by the two compounds. Inhibition of the CRM1 pathway by endogenously produced prostaglandin and/or exogenously applied 15d-PGJ(2) may contribute to its anti-inflammatory, anti-proliferative, and anti-viral effects.

Nup358 (also known as RanBP2), a component of the cytoplasmic filaments of the nuclear pore complex, has been implicated in various nucleocytoplasmic transport pathways. Here, we identify Nup358 as an important factor for transportin-mediated nuclear import. Depletion of Nup358 resulted in a strong inhibition of nuclear import of the human immunodeficiency virus type 1 (HIV-1) Rev protein. HIV-1 Rev is an RNA-binding protein that is required for CRM1 ( also known as exportin 1)-dependent nuclear export of unspliced or partially spliced viral RNA. We show that transportin is the major nuclear import receptor for HIV-1 Rev in HeLa cells. Overexpression of transportin strongly promoted nuclear import of HIV-1 Rev in Nup358-depleted cells, indicating that the import receptor becomes rate-limiting under these conditions. Importantly, the import rate of other transportin-dependent proteins was also significantly reduced in Nup358-depleted cells. Our data therefore suggest a general role for Nup358 in transportin-mediated nuclear import.

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structurefunction analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin a/beta-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo- and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.

Translocation of transport complexes across the nuclear envelope is mediated by nucleoporins, proteins of the nuclear pore complex that contain phenylalanine-glycine (FG) repeats as a characteristic binding motif for transport receptors. CRM1 (exportin 1), the major export receptor, forms trimeric complexes with RanGTP and proteins containing nuclear export sequences (NESs). We analyzed the role of the nucleoporin-like protein 1, NLP1 (also known as hCG1 and NUPL2) in CRM1-dependent nuclear transport. NLP1, which contains many FG repeats, localizes to the nuclear envelope and could also be mobile within the nucleus. It promotes the formation of complexes containing CRM1 and RanGTP, with or without NES-containing cargo proteins, that can be dissociated by RanBP1 and/or the cytoplasmic nucleoporin Nup214. The FG repeats of NLP1 do not play a major role in CRM1 binding. Overexpression of NLP1 promotes CRM1-dependent export of certain cargos, whereas its depletion by small interfering RNAs leads to reduced export rates. Thus, NLP1 functions as an accessory factor in CRM1-dependent nuclear protein export.