Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Structural Basis of High‐Affinity Nuclear Localization Signal Interactions with Importin‐α

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10–12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin‐α. The cNLSs bind along a concave groove on importin‐α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin‐α binding to both designed and naturally occurring high‐affinity cNLS‐like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap‐binding protein 80. Our data suggest that cNLSs and cNLS‐like sequences can achieve high affinity through maximizing interactions at the importin‐α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin‐α surface, and also expands on recent observations that longer linker sequences are allowed, and that long‐range electrostatic complementarity can contribute to cNLS‐binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.     

Nuclear Import of Chromatin Remodeler Isw1 Is Mediated by Atypical Bipartite cNLS and Classical Import Pathway

The protein Isw1 of Saccharomyces cerevisiae is an imitation‐switch chromatin‐remodeling factor. We studied the mechanisms of its nuclear import and found that the nuclear localization signal (NLS) mediating the transport of Isw1 into the nucleus is located at the end of the C‐terminus of the protein (aa1079–1105). We show that it is an atypical bipartite signal with an unconventional linker of 19 aa (KRIR X₁₉ KKAK) and the only nuclear targeting signal within the Isw1 molecule. The efficiency of Isw1 nuclear import was found to be modulated by changes to the amino acid composition in the vicinity of the KRIR motif, but not by the linker length. Live‐cell imaging of various karyopherin mutants and in vitro binding assays of Isw1NLS to importin‐α revealed that the nuclear translocation of Isw1 is mediated by the classical import pathway. Analogous motifs to Isw1NLS are highly conserved in Isw1 homologues of other yeast species, and putative bipartite cNLS were identified in silico at the end of the C‐termini of imitation switch (ISWI) proteins from higher eukaryotes. We suggest that the C‐termini of the ISWI family proteins play an important role in their nuclear import.     

KPNB1, XPO7 and IPO8 Mediate the Translocation ofNF‐κB/p65 into the Nucleus

NF‐κB/p65 is retained in the cytoplasm until it is activated in response to stress. Nuclear import of p65 is regulated by importin α in a nuclear localization signal (NLS)‐dependent manner. However, the role of importin β family members in the nuclear translocation of p65 is largely unclear. In this study, using high‐content siRNA screening, we identified three of 17 importin β family members that are involved in the nuclear import of p65. Our data showed that knockdown of KPNB1, XPO7 and IPO8 reduced the amount of nuclear p65 following tumor necrosis factor‐α (TNF‐α) stimulation, resulting in lower NF‐κB activity. KPNB1 was the major importin β receptor for p65 import, and this import was dependent on the NLS of p65. However, NLS‐mutated p65 still entered the nucleus and bound to XPO7 and IPO8. Interestingly, among the six members of the importin α family, KPNA2 was most important for p65 import. Taken together, our results show that the import of p65 mainly relies on the canonical KPNA2/KPNB1 pathway; however, p65 is also imported by an alternative pathway that is independent of its NLS. Redundant importin receptors are likely to maintain the important function of p65 according to need .      

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best‐characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin‐α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9‐12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.     

No discrete complexes containing DNA polymerase α activity can be solubilized from the heat-stabilized nuclear matrix prepared from HeLa S3 cells

Most of the DNA polymerase α activity, bound to the heat-stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5–20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix-bound DNA polymerase α activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37°C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase α to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.     

Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/β. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (−β, −7, −β/7, −13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Eukaryotic DNA primase abortive synthesis of oligoadenylates

Calf thymus DNA polymerase alpha-primase, human placenta DNA polymerase alpha-primase and human placenta DNA primase synthesized oligoriboadenylates of a preferred length of 2-10 nucleotides and multimeric oligoribonucleotides of a modal length of about 10 monomers on a poly(dT) template. The dimer and trimer were the prevalent products of the polymerization reaction. However, only the oligonucleotides from heptamers to decamers were elongated efficiently by DNA polymerase alpha.     

DNA repair in ultraviolet-irradiated HeLa cells is disrupted by aphidicolin: The inhibition of repair need not imply the absence of repair synthesis

Aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase α, has been reported to inhibit repair DNA synthesis in ultraviolet-irradiated, normal human fibroblasts but not in HeLa cells. By the use of assays for repair other than the measurement of repair synthesis, it is shown here that repair in HeLa cells is in fact susceptible to aphidicolin. Severe inhibition of DNA repair, with failure of individual repair events to be completed, and a smaller number of lesions removed, can occur even though repair synthesis continues.     

Catalytic properties of DNA polymerase α activity associated with the heat-stabilized nuclear matrix prepared from HeLa S3 cells

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH₄)₂SO₄ and led us to consider that a 37° incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.     

Distinctive Conformation of Minor Site‐Specific Nuclear Localization Signals Bound to Importin‐α

Nuclear localization signals (NLSs) contain one or two clusters of basic residues and are recognized by the import receptor importin‐α. There are two NLS‐binding sites (major and minor) on importin‐α and the major NLS‐binding site is considered to be the primary binding site. Here, we used crystallographic and biochemical methods to investigate the binding between importin‐α and predicted ‘minor site‐specific’ NLSs: four peptide library‐derived peptides, and the NLS from mouse RNA helicase II/Guα. The crystal structures reveal that these atypical NLSs indeed preferentially bind to the minor NLS‐binding site. Unlike previously characterized NLSs, the C‐terminal residues of these NLSs form an α‐helical turn, stabilized by internal H‐bond and cation‐π interactions between the aromatic residues from the NLSs and the positively charged residues from importin‐α. This helical turn sterically hinders binding at the major NLS‐binding site, explaining the minor‐site preference. Our data suggest the sequence RXXKR[K/X][F/Y/W]XXAF as the optimal minor NLS‐binding site‐specific motif, which may help identify novel proteins with atypical NLSs .     

The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair

The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.     

Ubiquitin ligase ARF‐BP1/Mule modulates base excision repair

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady‐state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF‐BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase β (Pol β), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol β and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol β and increased DNA repair. Our findings provide a novel mechanism regulating steady‐state levels of BER proteins.     

DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

Structure prediction and fold recognition for the ferrochelatase family of proteins

An α/β barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley-Liss Inc.