RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import

Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A > 400-kD complex contains p97, Ran, and RanBP2. Another complex of 150-300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of Ran-GDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitonin-permeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97.     

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.     

A distinct and parallel pathway for the nuclear import of an mRNA-binding protein

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.     

In vitro characterization of rice importin beta1: molecular interaction with nuclear transport factors and mediation of nuclear protein import

We recently isolated two cDNAs encoding importin 3 homologues (rice importin beta1 and beta2), the first such homologues identified in plants. To address the function of rice importin beta1 in the process of nuclear import of proteins, we carried out in vitro binding and nuclear import assays. Recombinant protein of rice importin beta1 assembled a complex (PTAC) with rice importin alpha1 and NLS protein, and also bound to the nuclear envelope of tobacco BY-2 cells. Ran-GTP, but not Ran-GDP, interacted with rice importin beta1 and dissociated the heterodimer formed between rice importin alpha1 and rice importin beta1. An in vitro nuclear import assay using digitonin-permeabilized HeLa cells revealed that rice importin beta1 can mediate nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data strongly suggest that rice importin beta1 functions as a component of the NLS receptor in plant cells.     

Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells

Here, we describe an application of the backbone cyclic (BC) proteinomimetic approach to the design and the synthesis of a BC peptide which functionally mimics the nuclear localization signal (NLS) region of the human immunodeficiency virus type 1 matrix protein (HIV-1 MA). On the basis of the NMR structure of HIV-1 MA, a library of BC peptides was designed and screened for the ability to inhibit nuclear import of NLS-BSA in digitonin-permeabilized HeLa and Colo-205 cultured cells. The screening yielded a lead compound (IC50 = 3 microM) which was used for the design of a second library. This library led to the discovery of a highly potent BC peptide, designated BCvir, with an IC50 value of 35 nM. This inhibitory potency is compared to a value of 12 microM exhibited by the linear parent HIV-1 MA NLS peptide. BCvir also reduced HIV-1 production by 75% in infected nondividing cultured human T-cells and was relatively resistant to tryptic digestion. These properties make BCvir a potential candidate for the development of a novel class of antiviral drugs which will be based on blocking nuclear import of viral genomes.     

A single mutation Asp229 --> Ser confers upon Gs alpha the ability to interact with regulators of G protein signaling

RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for Gi and Gq families of heterotrimeric G proteins but have not been found to interact with Gs alpha. The Gs alpha residue Asp229 has been suggested to be responsible for the inability of RGS proteins to interact with Gs alpha [Natochin, M., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 4300-4303]. To test this hypothesis, we have investigated the possibility of generating an interaction between Gs alpha and RGS proteins by substituting Gs alpha Asp229 with Ser and replacing the potential Gs alpha Asp229 contact residues in RGS16, Glu129 and Asn131, by Ala and Ser, respectively. RGS16 and its mutants failed to interact with Gs alpha. A single mutation of Gs alpha, Asp229Ser, rendered the Gs alpha subunit with the ability to interact with RGS16 and RGS4. Like RGS protein binding to Gi and Gq alpha-subunits, RGS16 preferentially recognized the AlF4--bound conformation of Gs alpha Asp229Ser. In a single-turnover assay, RGS16 maximally stimulated GTPase activity of Gs alpha Asp229Ser by approximately 5-fold with an EC50 value of 7.5 microM. Our findings demonstrate that Asp229 of Gs alpha represents a major barrier for Gs alpha interaction with known RGS proteins.     

A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light

A mouse gene, mper1, having all the properties expected of a circadian clock gene, was reported recently. This gene is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN). mper1 maintains this pattern of circadian expression in constant darkness and can be entrained to a new light/dark cycle. Here we report the isolation of a second mammalian gene, mper2, which also has these properties and greater homology to Drosophila period. Expression of mper1 and mper2 is overlapping but asynchronous by 4 hr. mper1, unlike period and mper2, is expressed rapidly after exposure to light at CT22. It appears that mper1 is the pacemaker component which responds to light and thus mediates photic entrainment.     

Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis

T helper cell (Th) 1, but not Th2, effectors undergo rapid Fas/Fas ligand (FasL)-mediated, activation-induced cell death upon restimulation with antigen. Unequal apoptosis is also observed without restimulation, after a longer lag period. Both effectors undergo delayed apoptosis induced by a non-Fas-mediated pathway. When Th1 and Th2 effectors are co-cultured, Th2 effectors survive preferentially, suggesting the responsible factor(s) is intrinsic to each population. Both Th1 and Th2 effectors express Fas and FasL, but only Th2 effectors express high levels of FAP-1, a Fas-associated phosphatase that may act to inhibit Fas signaling. The rapid death of Th1 effectors leading to selective Th2 survival provides a novel mechanism for differential regulation of the two subsets.     

Association among p53 gene mutation, nuclear accumulation of the p53 protein and aggressive phenotypes in breast cancer

In order to reveal whether differences in the type and site of p53 gene mutations influence the function of the gene and tumor phenotype, we examined nuclear accumulation of the p53 protein immunohistochemically, loss of the other p53 allele by restriction-fragment-length polymorphism analysis, and histological grade of atypia in 52 breast-cancer tissue specimens in which the position and pattern of the mutation were identified. When mis-sense point mutations or deletions of 3n bases (n = 1, 2, ...), which did not cause a frameshift downstream, occurred within codons 110-180 or 234-285, containing highly conserved regions, the p53 protein was almost always (92%) accumulated in nuclei in a majority of the cancer cells. When these mutations occurred outside these regions, only 46% of the cases showed nuclear accumulation of the protein in a majority of cancer cells. In tumors with non-sense point mutations or deletion of 3n + 1 or 3n + 2 bases (n = 0, 1, 2, ...), which caused a downstream frameshift, nuclear accumulation of the p53 protein was absent in 93% of cases. Irrespective of the mutation site or pattern, a majority of cases showing p53 mutation revealed loss of heterozygosity on 17p13 (83%), which suggested they do not carry wild-type p53 allele, and the highest histological grade of atypia (90%). Regardless of differences in their protein-expression pattern, a majority of the p53 gene mutations were suggested to function in a recessive mode and to be involved in the development of histologically aggressive breast cancer.     

A novel F box protein, NFB42, is highly enriched in neurons and induces growth arrest

NFB42 (neural F Box 42 kDa) is a novel gene product that is highly enriched in the nervous system. Its predicted protein contains an F box, a motif recently shown to couple cell cycle regulation to the proteasome pathway (Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J. W., and Elledge, S. (1996) Cell 86, 263-274). NFB42 mRNA and protein are expressed in all major areas of the adult rat brain but are not detected in non-neural tissues. NFB42 protein is localized primarily to the cytoplasm of neurons and does not appear to be present in glia. The presence of an F box in NFB42 suggests that it may be involved in cell cycle regulation; however, its expression in postmitotic neurons indicates that it is not involved in regulating typical cell cycle events. In an initial attempt to characterize the function of this protein, NFB42 was transfected into N1E-115 neuroblastoma and Chinese hamster ovary cells. The expression of full-length NFB42, but not an F box deletion mutant, inhibits proliferation in both cell lines. Additional experiments demonstrate that NFB42 interacts with Skp1p, a component of the proteasome pathway, and deletion of the F box also inhibits this interaction. Overall, the expression pattern of NFB42, along with the presence of an F box domain and the ability to inhibit growth, suggests that it may play a role in maintaining neurons in a postmitotic state.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells

The assembly of eukaryotic ribosomal subunits takes place in the nucleolus and requires nuclear import of ribosomal proteins. We have studied this import in a mammalian system and found that the classical nuclear import pathway using the importin alpha/beta heterodimer apparently plays only a minor role. Instead, at least four importin beta-like transport receptors, namely importin beta itself, transportin, RanBP5 and RanBP7, directly bind and import ribosomal proteins. We found that the ribosomal proteins L23a, S7 and L5 can each be imported alternatively by any of the four receptors. We have studied rpL23a in detail and identified a very basic region to which each of the four import receptors bind avidly. This domain might be considered as an archetypal import signal that evolved before import receptors diverged in evolution. The presence of distinct binding sites for rpL23a and the M9 import signal in transportin, and for rpL23a and importin alpha in importin beta might explain how a single receptor can recognize very different import signals.     

Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for staphylococcal protein A and streptococcal protein G

Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purification of the subisotypes of equine IgG. Purification of IgGa and IgGb was achieved by the separation of a 'fall-through' peak from ion-exchange chromatography consisting of IgGa and IgGb into two fractions (peaks C and D) by FPLC protein A and protein G affinity chromatography. Peak C consisted of IgGb and peak D consisted of IgGa exhibiting slightly faster cathodal migration than peak C in IEP analysis. Affinity chromatography using protein A and G columns also indicated that there may be two different components of IgG(T); one with a low affinity for protein G and the other having a greater affinity for protein G.     

A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue

We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.     

A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.