Karyopherin α2 is a maternal effect gene required for early embryonic development and female fertility in mice.

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene.

Kpna6 deficiency causes infertility in male mice by disrupting spermatogenesis.

Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The karyopherin α family of proteins mediates movement of specific cargo proteins when bound to karyopherin ß. Karyopherin α genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here, we use a loss-of-function approach to determine specifically the role of Kpna6 in spermatogenesis and male fertility. We show that ablation of Kpna6 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Kpna6-deficient mice exhibit impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear localization of the androgen receptor. Furthermore, our data demonstrate devastating defects on spermiogenesis, including incomplete sperm maturation and a massive reduction in sperm number, accompanied by disturbed histone-protamine exchange, differential localization of the transcriptional regulator BRWD1 and altered expression of RFX2 target genes. Our work uncovers an essential role of Kpna6 in spermatogenesis and, hence, in male fertility.

A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, endoplasmic reticulum lectin 1 (ERLEC1), inactive tyrosine-protein kinase 7 (PTK7), and DnaJ homolog subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences.

Preprotein signature for full susceptibility to the co-translational translocation inhibitor cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor.

Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide.

One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates.

Karyopherin α-3 is a key protein in the pathogenesis of spinocerebellar ataxia type 3 controlling the nuclear localization of ataxin-3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3.

A hyperactive mutant of interferon-regulatory factor 4.

We found that deletion of the final 30 amino acids of transcription factor IRF4's (interferon-regulatory factor) C-terminus creates hyperactive IRF4. When introduced into IRF4-deficient CD4+ or CD8+ T cells, more type 17 differentiation was found compared to WT IRF4. Interestingly, Th9 differentiation and Th2-linked IL-13 production were much less altered.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons.

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin ß1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.

Signal peptide-binding drug as a selective inhibitor of co-translational protein translocation.

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.

Identification of importin α 7 specific transport cargoes using a proteomic screening approach.

The importin α:ß complex is responsible for the nuclear import of proteins bearing classical nuclear localization signals. In mammals, several importin α subtypes are known to exist that are suggested to have individual functions. Importin α 7 was shown to play a crucial role in early embryonic development in mice. Embryos from importin α 7-depleted females stop at the two-cell stage and show disturbed zygotic genome activation. As there is evidence that individual importin α subtypes possess cargo specificities, we hypothesized that importin α 7 binds a unique set of intracellular proteins. With the use of a collection of in vitro and in vivo binding assays, importin α 7 interaction partners were identified that differed from proteins found to bind to importin α 2 and 3. One of the proteins preferentially binding importin α 7 was the maternal effect protein Brg1. However, Brg1 was localized in oocyte nuclei in importin α 7-deficient embryos, albeit in reduced amounts, suggesting additional modes of nuclear translocation of this factor. An additional SILAC-based screening approach identified Ash2l, Chd3, Mcm3, and Smarcc1, whose nuclear import seems to be disturbed in importin α 7-deficient fibroblasts.

Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice.

Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-α isoforms. Thereby, it blocks STAT1-mediated IFN-α/ß and IFN-γ synthesis. However, owing to the lack of importin-α knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-α7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-α7 was not sufficient to increase survival rates among mice infected with EBOV.

Single rRNA helices bind independently to the protein-conducting channel SecYEG.

Ribosomes tightly interact with protein-conducting channels in the plasma membrane of bacteria (SecYEG) and in the endoplasmic reticulum of eukaryotes (Sec61 complex). This interaction is mediated by multiple junctions and is highly conserved during evolution. Although it is well known that both ribosomal proteins and ribosomal RNA (rRNA) are involved in the ribosome-channel interaction, detailed analyses on how these components contribute to this binding are lacking. Here, we demonstrate that the evolutionary conservation of ribosome binding is solely mediated by rRNA. Moreover, we show that in vitro transcribed 23 S rRNA binds with similar characteristics to protein translocation channels as native 23 S rRNA or 50 S ribosomal subunits. This indicates that base modifications, which exist in native rRNA, do not crucially influence the binding. In two of the ribosome-channel junctions (c1 and c2), exclusively rRNA helices are involved. Using in vitro transcribed rRNA mutants, we now provide evidence that large parts of the rRNA can be deleted without altering its binding properties, as long as the rRNA helices of the c1 and c2 junctions remain intact. We demonstrate that the connection sites c1 and c2 generate high-affinity binding sites that act independently of each other. This could explain why membrane-bound ribosomes have an extremely low off-rate.

Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation.

Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes. Thus, RNase-neutralized pancreatic membrane fractions from pig, cow, or sheep are a cheap, easily accessible, and fulfilling alternative to canine microsomes.

TRAP assists membrane protein topogenesis at the mammalian ER membrane.

Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the 'positive-inside' rule.

High-throughput sequencing of a single chromosome: a moth W chromosome.

Y and W chromosomes have mostly been excluded from whole genome sequencing projects. Due to the high amount of repetitive sequences they are 'difficult' to assemble and therefore need special treatment in the form of, e.g. adapted assembly programs, a range of different libraries, and accurate maps, if possible. A minimum requirement for these approaches is pure template DNA. We therefore microdissected the W chromatin of highly polyploid cells from the flour moth, Ephestia kuehniella, and used Roche/454 and Sanger sequencing to generate 72.6 Mbp of DNA sequence. Nominal coverage was 4.3× of the 16.7 Mbp of W chromosomal DNA. We used these data to assess the genetic content of the W chromosome. This approach allowed us to determine constituent families of transposable elements, microsatellites, and recent insertion sites of mitochondrial DNA. However, no conventional protein-coding gene has yet been found. The sequence collection is a rich source for the definition of W-specific PCR markers and the reconstruction of W chromosome loci, as a step towards full reconstruction of the chromosome.

EcmPred: prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection.

The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred.

Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative.

During cotranslational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the endoplasmic reticulum (ER) membrane. Our cryo-electron microscopy structure of ribosome-Sec61 shows binding of an ordered heterotetrameric translocon-associated protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28S ribosomal RNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, ß, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-α/ß/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the cyclotriazadisulfonamide derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147 resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.

Data on draft genomes and transcriptomes from females and males of the flour moth, Ephestia kuehniella.

We present genomes and pupal transcriptomes of the Mediterranean flour moth, Ephestia kuehniella. The moth is a world-wide storage pest as well as a laboratory species with a considerable background in developmental biology, genetics, and cytogenetics. The sequence data were derived from a highly inbred laboratory strain and, hence, display very little heterozygosity. Female and male genomes and transcriptomes are represented separately in two sets each of raw and assembled sequence data. They are designed as a basis to develop new strategies in pest control, to elucidate the molecular adaptation for its peculiar lifestyle, and for research on sex chromosome structure, sex determination and sex-specific gene activity. For a test, all genes known or suspected to have a role in sex determination were extracted from the data. Raw sequencing data and assemblies are available at European Nucleotide Archive under accession number PRJEB49052.

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes.

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.

Tdrd3 is a novel stress granule-associated protein interacting with the Fragile-X syndrome protein FMRP.

Tudor domains are widespread among proteins involved in RNA metabolism, but only in a few cases their cellular function has been analyzed in detail. Here, we report on the characterization of the ubiquitously expressed Tudor domain containing protein Tdrd3. Apart from its Tudor domain, we show that Tdrd3 possesses an oligosaccharide/nucleotide binding fold (OB-fold) and an ubiquitin associated domain capable of binding tetra-ubiquitin. A set of biochemical experiments revealed an interaction of Tdrd3 with FMRP, the product of the gene affected in Fragile X syndrome, and its autosomal homologs FXR1 and FXR2. FMRP has been implicated in the translational regulation of target mRNAs and shown to be a component of stress granules (SG). We demonstrate that overexpression of Tdrd3 in cells induces the formation of SGs and as a result leads to its co-localization with endogenous FMRP in these structures. Interestingly, the disease-associated FMRP missense mutation I304N identified in a Fragile X patient severely impairs the interaction with Tdrd3 in biochemical experiments. We propose a contribution of Tdrd3 to FMRP-mediated translational repression and suggest that the loss of the FMRP-Tdrd3 interaction caused by the I304N mutation might contribute to the pathogenesis of Fragile X syndrome.