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How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron–specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3–bound c-Fos and importin α3–deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.

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 β. 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., Summary: Two different mouse models delineate the morphological and functional impact of Kpna6 on spermatogenesis and Sertoli cell function and show that this protein is crucial for fertility in male mice.

Summary Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections., Graphical Abstract, Highlights • Importin-α3 is the most abundantly expressed isoform in the mammalian lung • Importin-α3 is highly conserved across species • Importin-α3 is one of the major nuclear transporters of NF-κB • Importin-α3 acts as an immune sensor of influenza A virus infections, Thiele et al. show that importin-α3 is one of the major nuclear transporters of NF-κB in the mammalian lung. High-level TNF-α-inducing HPAIVs inhibit importin-α3 mRNA transcription by interfering with its promoter activity. Thus, HPAIVs may evade antiviral immunity in the respiratory tract by generating a bottleneck in importin-α3 availability.

Summary Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons., Graphical Abstract, Highlights • Reduced expression of importin α5 in hippocampal neurons decreases anxiety • Importin α5 is required for nuclear localization of MeCP2 in hippocampal neurons • Importin α5 knockout increases expression of Sphk1, an MeCP2-regulated gene • Pharmacological modulation of Sphk1 and the S1P receptor affects anxiety, Panayotis et al. found decreased anxiety in importin α5 knockout mice. They report that importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.

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.

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.

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.

During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61α-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-{alpha} isoforms. Thereby, it blocks STAT1-mediated IFN-{alpha}/{beta} and IFN-{gamma} synthesis. However, owing to the lack of importin-{alpha} knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-{alpha}7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-{alpha}7 was not sufficient to increase survival rates among mice infected with EBOV.

The 5'-end maturation of tRNAs is catalyzed by the ribonucleoprotein enzyme ribonuclease P (RNase P) in all organisms. Here we provide, for the first time, a comprehensive overview on the representation of individual RNase P protein homologs within the Eukarya and Archaea. Most eukaryotes have homologs for all four protein subunits (Pop4, Rpp1, Pop5 and Rpr2) present in the majority of Archaea. Pop4 is the only RNase P protein subunit identifiable in all Eukarya and Archaea with available genome sequences. Remarkably, there is no structural homology between bacterial and archaeal-eukaryotic RNase P proteins. The simplest interpretation is that RNase P has an 'RNA-alone' origin and progenitors of Bacteria and Archaea diverged very early in evolution and then pursued completely different strategies in the recruitment of protein subunits during the transition from the 'RNA-alone' to the 'RNA-protein' state of the enzyme.

SMNrp, also termed SPF30, has recently been identified in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP-associated protein that appears in the pre-spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the first step of pre-mRNA splicing by preventing the formation of complex B. Re-addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6⋅U5] tri-snRNP. A candidate that may mediate this interaction, namely the U4/U6-90 kDa protein, has been identified. We suggest that SMNrp, as a U2 snRNP-associated protein, facilitates the recruitment of the [U4/U6⋅U5] tri-snRNP to the pre-spliceosome.

Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66–amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61α and Sec61β, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61α and Sec61β, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.

Nuclear proteins like transcription factors and ribosomal proteins are synthesized in the cytoplasm and have to be transported into the nucleus to fulfill their functions. The transport of proteins >20–60 kD through the nuclear pore complex (NPC) into the nucleus is an active, energy-requiring process. Transport substrates are recognized by their transport proteins via certain signals. The best-characterized protein import pathway is the ‘classical’ nuclear localization signal-dependent pathway with importin α and β carrying the substrate to the NPC. The transport of the importin-substrate complex into the nucleus is regulated by the small GTPase Ran/TC4. During the last years more than ten proteins have been discovered which have already been proven or are very likely to be nuclear transport factors of distinct import pathways: members of the importin α protein family are very similar and transport in complex with importin β nuclear localization signal-bearing proteins into the nucleus. Members of the Ran-binding protein family show some weak similarity to importin β. Sharing a common domain at the amino terminus, they are able to bind RanGTP, a prerequisite for their function as nuclear import or export factors for distinct proteins or RNAs. However, Ran/TC4 seems to play a key regulatory role in all nuclear transport pathways described so far, although the molecular mechanism of the translocation step through the NPC is still unclear.

In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.

The import of many proteins into the nucleus is mediated by the importin-alpha/beta heterodimer. While only one importin-beta gene has been found, several forms of importin-alpha have been described. In addition to the three human importin-alphas already identified, we report here the primary structure of two new human importin-alpha proteins. The five known human importin-alpha subunits can be classified into three subfamilies that appear conserved in higher eukaryotic organisms. We show by immunoblotting that the different importin-alpha subfamilies are expressed in a variety of human tissues and mammalian cell lines.

Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.

Background: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60 kD importin protein (importin 60) as a single polypeptide. Results We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)–nitrolotriacetic acid–Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin–agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. Conclusion Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.

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.

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.

Protein translocation into the mammalian endoplasmic reticulum requires the Sec61p complex, which consists of three membrane proteins. The alpha-subunit, the homologue of Sec61p of yeast, shows some similarity to SecYp, a key component of the protein export apparatus of bacteria. In Escherichia coli, SecYp is also associated with two other proteins (SecEp and band-1 protein). We have now determined the sequences of the beta- and gamma-subunits of the mammalian Sec61p complex. Sec61-gamma is homologous to SSS1p, a suppressor of sec61 mutants in Saccharomyces cerevisiae, and can functionally replace it in yeast cells. Moreover, Sec61-gamma and SSS1p are structurally related to SecEp of E. coli and to putative homologues in various other bacteria. At least two subunits of the Sec61/SecYp complex therefore seem to be key components of the protein translocation apparatus in all classes of organisms.

Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes.

Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-alpha subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin alpha/beta dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of "classical" importin alpha/beta receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.

Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane.

Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1−/− mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1−/− mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1−/− mice, and growth hormone production was slowed in SERP1−/− pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1−/− pancreas. In basal conditions, the Sec61 complex in SERP1−/− microsomes was more cofractionated with ribosomes, compared with SERP1+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.

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 (see Stavru et al. on p. 477 of this issue), 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.

THE import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs)1,2, energy3,4 and soluble factors5, namely importin-α(Mr 60K)6-12,28, importin-β (90K)8-11,13 and Ran14,15. Importin-α is primarily responsible for NLS recognition6-12,29 and is a member of a protein family that includes the essential yeast nuclear pore protein SRPlp (ref. 16). As the first event, the complex of importin-α and importin-β binds the import substrate in the cytosol8,9. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-β. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-α in the nucleus. In contrast, importin-β accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-β on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin beta-superfamily. Here we identify exportin 6 (Exp6) as a novel family member from higher eukaryotes and show that it mediates nuclear export of profilin.actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only approximately 20% identical amino acid residues, their function in profilin.actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. In contrast to a previous report, we found no indications of a major and direct role for CRM1 in actin export from mammalian or insect nuclei.

Importin beta-type transport receptors mediate the vast majority of transport pathways between cell nucleus and cytoplasm. We identify here the translation elongation factor 1A (eEF1A) as the predominant nuclear export substrate of RanBP21/exportin 5 (Exp5). This cargo-exportin interaction is rather un usual in that eEF1A binds the exportin not directly, but instead via aminoacylated tRNAs. Exp5 thus represents the second directly RNA-binding exportin and mediates tRNA export in parallel with exportin-t. It was suggested recently that 10-15% of the cellular translation would occur in the nucleus. Our data rule out such a scenario and instead suggest that nuclear translation is actively suppressed by the nuclear export machinery. We found that the vast majority of translation initiation factors (eIF2, eIF2B, eIF3, eIF4A1, eIF5 and eIF5B), all three elongation factors (eEF1A, eEF1B and eEF2) and the termination factor eRF1 are strictly excluded from nuclei. Besides Exp5 and importin 13, CRM1 and as yet unidentified exportins also contribute to the depletion of translation factors from nuclei.

We recently cloned six human importin a proteins that transport specific substrates in complex with importin beta into the nucleus. We now compared their absolute expression levels in different human cell lines. We examined their expression regulation during human cell proliferation and differentiation by means of specific antibodies. Proliferation inhibition by starvation of HeLa and HaCaT cells led to a marked decrease in the expression of various nuclear transport factors. In contrast, re-addition of serum increased alpha-importin expression. We analyzed two models for cell differentiation and found differential importin regulation. Stimulation of rat pancreatic AR42J cell differentiation towards a neuroendocrine phenotype with activin A or towards an acinar phenotype with dexamethasone, caused strong upregulation of importin alpha3 and alpha4 expression. Phorbol ester-induced differentiation of human leukemia (HL60) cells towards a macrophage phenotype led to downregulation of importin alpha1 and alpha4 expression after 72 hours. Similarly, importins alpha1 and alpha4 displayed a strong downregulation when HL60 cells were directed towards a neutrophil phenotype by DMSO treatment. This study is the first to assess all the human importin alpha isoforms in documenting differential nuclear transport factor regulation during cell proliferation and differentiation.

Increased importin α protein expression in diabetic nephropathy. Background Importins transport kinases, transcription factors, and viral proteins into the nucleus. Since the expression of several genes is increased in diabetic nephropathy, we tested the hypothesis that importin protein expression is increased in diabetic kidneys. Methods Immunohistochemistry and Western blotting were used in kidneys from streptozotocin-treated diabetic rats and from spontaneously diabetic Goto-Kakizaki rats. The effects of high glucose and mannose also were tested in cell culture experiments. Results In normal rat kidneys, importin α isoforms were differentially expressed in glomerular cells and tubular segments, while importin α1/Rch1 was expressed only in tubules and peritubular cells. In diabetic rat kidneys from both models, the importin α isoform expression was markedly up-regulated. Western blotting revealed strong up-regulation of importin α7 and minor up-regulation of other isoforms. Exposure of various cell types to high glucose or mannose (25 mmol/L) led to increased expression of importins α3, α5/hSRP1, and α7 in different cultured cells, while up-regulation of other importin α isoforms was less consistent. Conclusions A specific importin α isoform up-regulation takes place in kidneys of diabetic rats. Diabetes is a stimulus for increased importin α7 expression. Thus, nuclear transport in diabetes may be increased in glomerular and tubular cells. The signaling pathways appear differentially regulated in glomeruli, proximal, and distal tubules. The enhanced nuclear transport may participate in increased gene expression and nephrosclerosis in diabetes.

Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

Two subunits of the mammalian signal peptidase complex, SPC12 and SPC25, share similar membrane topologies with the majority of each protein oriented toward the cytoplasm. Such similarities may suggest that these proteins perform redundant functions in signal peptidase activity. In the present study, we addressed this issue through analysis of the yeast homologs to SPC12 and SPC25, Spc1p and Spc2p. We show that both Spc1p and Spc2p are nonessential for signal peptidase activity and growth of yeast cells and that null mutations in the genes encoding Spc1p and Spc2p are synthetically lethal with a conditional mutation affecting Sec11p, an essential subunit of yeast signal peptidase. However, a high copy plasmid encoding Spc1p suppresses the conditional sec11 mutation, whereas the corresponding plasmid encoding Spc2p does not suppress sec11. Moreover, Spc2p, but not Spc1p, is important for signal peptidase activity and cell viability at high temperatures. These results indicate that although both Spc1p and Spc2p are noncatalytic, they are functionally distinct. Evidence is also presented that a double mutant lacking Spc1p and Spc2p grows well relative to wild type yeast cells, indicating that the signal peptidase complex missing at least two of its subunits is sufficient for signal peptidase activity in vivo.

Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.

The complex of importin-alpha and -beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin-alpha provides the nuclear localization signal binding site, importin-beta the site of initial docking to the pore. Here we show that the conserved, basic N-terminus of importin-alpha is sufficient for importin-beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full-length importin-alpha itself. Transport is dependent on importin-beta but competed by importin-alpha. As no additional part of importin-alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin-beta and structures of the nuclear pore. The domain that binds to importin-beta appears to confer import only, but not re-export out of the nucleus, suggesting that the return of importin-alpha into the cytoplasm is not a simple reversal of its entry.

The alpha-subunit of the TRAP complex (TRAP alpha) is a single-spanning membrane protein of the endoplasmic reticulum (ER) which is found in proximity of nascent polypeptide chains translocating across the membrane. Here, we demonstrate the widespread occurrence of TRAP alpha in eukaryotes as indicated by its existence in man, fish and plants. Despite the fact that the sequence homology is much lower than for other proteins in the translocation site, the overall topology, the location of the glycosylation sites and, most interestingly, the distribution of charges are conserved. These data indicate that the TRAP complex has a ubiquitous function.

A chemically charged amber suppressor tRNA was used to introduce the photoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed the interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal sequence to be investigated during insertion into the membrane of the endoplasmic reticulum (ER). We found that different regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membrane protein) contacts the NH2-terminal region of the signal sequence while the mammalian Sec61p contacts the hydrophobic core of the signal sequence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subunits which contact distinct regions of nascent polypeptides during their membrane insertion.

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called 'signal sequence receptor' that is now renamed as 'translocon-associated protein' (TRAP). Two glycosylated subunits of the TRAP complex have been identified before (alpha and beta subunits). We now show that TRAP complex is actually comprised of four membrane proteins (alpha, beta, gamma, delta), present in a stoichiometric relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, non-glycosylated subunits were deduced from cloning of the corresponding cDNAs. The delta subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The gamma subunit is predicted to span the membrane four times.

SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.

To identify components of the mammalian endoplasmic reticulum involved in the translocation of secretory proteins, crosslinking and reconstitution methods were combined. A multispanning abundant membrane glycoprotein was found which is in proximity to nascent chains early in translocation. In reconstituted proteoliposomes, this protein is stimulatory or required for the translocation of secretory proteins.

The signal-sequence receptor (SSR) has previously been shown to be a component of the environment which nascent polypeptides meet on passage through the endoplasmic reticulum (ER) membrane. We report here on the primary structure of the SSR as deduced from cDNA clones and from direct protein sequencing. The glycoprotein is synthesized with a cleavable amino-terminal signal sequence and contains only one classical membrane-spanning segment. Its insertion into the ER membrane during biosynthesis depends on the function of the signal-recognition particle. SSR shows a remarkable charge distribution with the amino terminus being highly negatively charged, and the cytoplasmic carboxyl terminus positively charged. The SSR can be phosphorylated in its cytoplasmic tail both in intact cells and in a cell-free system, suggesting a regulation of its function. The localization of the protein in the ER membrane was confirmed by immunofluorescence microscopy.

In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.

We have purified a glycosylated, membrane-spanning protein of relative molecular mass approximately 34,000 (Mr approximately 34 K) from canine microsomes that appears to be essential for protein translocation across the endoplasmic reticulum (ER) as shown by the inhibitory action of antibodies directed against it and of monovalent Fab-fragments produced from them. The ER membrane contains at least as many molecules of the 34 K membrane protein as bound ribosomes. The protein can be detected immunologically in tissues of various organisms, indicating an universal function.

We have developed a rule to predict the orientation of the first internal signal-anchor sequence in eukaryotic transmembrane proteins synthesized on the rough endoplasmic reticulum. The difference in the charges of the 15 residues flanking the first internal signal-anchor determines its orientation, with the more positive portion facing the cytosol. In proteins that span the membrane more than once, the orientation of all subsequent transmembrane segments would be determined by that of the most N-terminal one.

Photocrosslinking has been used to identify integral proteins of the endoplasmic reticulum membrane that are in proximity to nascent preprolactin during in vitro translocation. A photoreactive lysyl derivative was introduced into truncated preprolactin chains comprising 86 or 115 amino acids. Both with the 86mer, containing the reactive group in the signal sequence, and with the 115mer, containing the probe exclusively in the mature portion of the chain, photocrosslinking occurred to a 3̃5 kDa transmembrane glycoprotein, the signal sequence receptor (SSR). SSR is identical with a previously isolated abundant and ubiquitous 34 kDa membrane protein that appears to be essential for protein translocation.

Protein translocation across the endoplasmic reticulum (ER) membrane is triggered at several stages by information contained in the signal sequence. Initially, the signal sequence of a nascent secretory protein upon emergence from the ribosome is recognized by a polypeptide of relative molecular mass 54,000 (Mr 54K) which is part of the signal recognition particle (SRP)1,2,27. Binding of SRP may induce a site-specific elongation arrest of translation in vitro1,3. Attachment of the arrested translation complex to the ER membrane is mediated by the SRP-receptor (docking protein)3,4 and is accompanied by displacement of the SRP from both the ribosome5 and the signal sequence6. We have investigated the fate of the signal sequence following the disengagement of SRP and its receptor by a crosslinking approach2,6. We report here that the signal sequence of nascent preprolactin, after its release from the SRP, interacts with a newly discovered component, a signal sequence receptor (SSR), which is an integral, glycosylated pmrotein of the rough ER membrane (Mr ∼35K).