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 structure-function 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 α/β-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.

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin ␤, but not on the adapter protein importin ␣. We now show that, similar to importin ␣, Rev is able to dissociate RanGTP from recycling importin ␤, a reaction that leads to the formation of a novel import complex. Besides importin ␤, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin ␤-binding domain of importin ␣, Rev interacts with an N-terminal fragment of importin ␤. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin ␤ and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.The machinery for transport of macromolecules across the nuclear envelope consists of the nuclear pore complex, which is embedded between the inner and outer nuclear membrane (1), and a large variety of soluble transport factors (for reviews see Refs. 2-4). The transport cargoes are characterized by recognition sequences for soluble transport receptors: nuclear export sequences mediate the interaction of proteins with exportins, allowing transport out of the nucleus, whereas nuclear localization signals (NLSs) 2 bind to importins, promoting transport of proteins into the nucleus. Importins and exportins, collectively also referred to as karyopherins, belong to the importin ␤ superfamily of transport receptors. They interact not only with their transport cargo but also with proteins of the nuclear pore complex, as well as with the small GTP-binding protein Ran, a factor that plays an important role in the assembly or disassembly of transport complexes (3). Importin ␤, the prototype of this family, was originally identified as the transport receptor that is involved in nuclear import of proteins containing a "classic" NLS, i.e. a short stretch of amino acids enriched in basic residues, forming either a single or a bipartite transport motif. These NLSs do not bind to importin ␤ directly but via an adapter protein, importin ␣. Importin ␣ contains a characteristic importin ␤-binding-(IBB) domain. A ternary complex containing the import cargo, importin ␣, and importin ␤ assembles in the cytoplasm, translocates across the nuclear pore complex, and dissociates in the nucleus upon binding of RanGTP to importin ␤. The import receptor then recycles back to the cytoplasm in a complex with RanGTP. A dedicated exportin, CAS, is used for export of importin ␣ out of the nucleus (5). Hundreds of proteins...

The signaling molecule 15-deoxy-⌬ 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.

The mechanism of the antagonistic action of insulin on the glucagon-dependent stimulation of the phosphoe/i0/pyruvate carboxykinase (PEPCK) gene was studied in primary cultures of rat hepatocytes. Gene expression was monitored by the transcriptional activity of the PEPCK gene and the accumulation and degradation of PEPCK mRNA.

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon.1. The mRNA of PEPCK was induced maximally 2-3 h after addition of 10 nM glucagon, as detected by Northern-blot analysis after hybridization with a biotinylated antisense RNA of PEPCK.2. The synthesis rate of PEPCK increased maximally 2-3 h after application of glucagon as revealed by pansorbin-linked iinmunoprecipitation of [35SJmethionine-labelled PEPCK.3. The enzyme amount and activity was maximally induced 4 h after glucagon application. 4. The mRNA of PEPCK was half-maximally induced by 0.1 nM and maximally by I nM and 10 nM glucagon. The half-maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin.The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon-dependent increase in mRNA and enzymesynthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1 -1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range with glucagon being the dominant hormone.

c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin ␤ superfamily, importin ␤ itself and transportin, promote import of c-Fos in vitro. Under conditions where importin ␤-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin ␤ were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin ␤ for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin ␤ in recognizing different nuclear import cargoes.Import of proteins into the nucleus is mediated by importins, members of the importin ␤ superfamily. The "classic" signals for nuclear import (nuclear localization signal; NLS) 2 are short basic stretches of amino acids with lysines as characteristic components, which may occur either as a single or a bipartite motif. These classic NLSs are recognized by the adapter molecule importin ␣, which forms a heterodimer with the actual transport receptor, importin ␤ (for reviews see Refs. 1-3). The importin ␣/␤-NLS-cargo complex is then translocated through the nuclear pore complex. Nucleoporins, the protein components of the nuclear pore complex, may interact with the transport receptor, facilitating the translocation of the complex. Importin ␤ interaction with RanGTP on the nuclear side of the nuclear pore complex results in dissociation of the transport complex (4). Hundreds of individual nuclear proteins or proteins that shuttle between the nucleus and the cytoplasm are thought to use this classic importin ␣/␤ nuclear import pathway. Over the last couple of years, however, a number of proteins have been described that do not depend on the adapter protein importin ␣. Some of these bind directly to the classic import receptor, importin ␤, without requiring any adapter protein. These include the proteins Rev and Rex of the human immunodeficiency virus (5, 6) and the human T-cell leukemia virus (7), respectively, the T-cell protein tyrosine phosphatase (8), the parathyroid hormone-related protein (PTHrP) (9), cyclin B1 (10), the sterol regulatory element-binding protein 2 (SREBP-2 (11)), the zinc finger protein Snail (12), and the transcription factor CREB (13). Many of these proteins also contain basi...

The influence of recombinant human interleukin-6, the major mediator of the inflammatory response in liver, on the glucagon- and insulin-dependent induction of the phosphoenolpyruvate carboxykinase and glucokinase gene, respectively, was monitored on the level of gene transcription, mRNA abundance and enzyme activity in cultured rat hepatocytes. As control markers of the interleukin-6-induced acute-phase response the mRNA levels of the acute phase proteins alpha 2-macroglobulin and beta-fibrinogen were determined. In cultured rat hepatocytes, recombinant human interleukin-6, added simultaneously with glucagon and insulin, lowered the maximal increase in glucagon-induced phosphoenolpyruvate carboxykinase mRNA levels after 2 hr and the maximal increase in glucokinase mRNA levels after 3 hr to about 30%, respectively. It inhibited the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene transcription and phosphoenolpyruvate carboxykinase enzyme activity, as well as the insulin-induced increases in glucokinase gene transcription and glucokinase enzyme activity. Recombinant human interleukin-6 increased the mRNA levels of the acute-phase proteins alpha 2-macroglobulin and beta-fibrinogen gradually over 4 to 6 hr. Recombinant human interleukin-6, added 2 hr after glucagon or 3 hr after insulin at the maximum of the hormone-induced enzyme mRNA levels, almost doubled the decay rate of phosphoenolpyruvate carboxykinase mRNA and glucokinase mRNA. The results show that interleukin-6 induced the expression of inflammatory proteins and simultaneously inhibited the hormone-induced expression of enzymes of intermediary metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3′end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT.