Type Of Nuclear Localization Signal Research Articles

Multiple types of nuclear localization signals in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite that belongs to the Amoebozoa supergroup whose study related to the nucleocytoplasmic transport of proteins through the nucleus is poorly studied. In this work, we have performed in silico predictions of the potential nuclear localization signals (NLS) corresponding to the proteome of 8201 proteins from Entamoeba histolytica annotated in the AmoebaDB database. We have found the presence of monopartite nuclear localization signals (MNLSs), bipartite nuclear localization signals (BNLSs), and non-canonical monopartite NLSs with lengths exceeding 20 amino acid residues. Additionally, we detected a new type of NLS consisting of multiple juxtaposed bipartite NLSs (JNLSs) that have not been described in any eukaryotic organism. Also, we have generated consensus sequences for the nuclear import of proteins with the NLSs obtained. Docking experiments between EhImportin α and an MNLS, BNLS, and JNLS outlined the interacting residues between the Importin and cargo proteins, emphasizing their putative roles in nuclear import. By transfecting HA-tagged protein constructs, we assessed the nuclear localization of MNLS (U1A and U2AF1), JMNLS (U2AF2), and non-canonical NLS (N-terminus of Pol ll) in vivo. Our data provide the basis for understanding the nuclear transport process in E. histolytica.

WW domains form a folded type of nuclear localization signal to guide YAP1 nuclear import.

The nuclear translocation of YAP1 is significantly implicated in the proliferation, stemness, and metastasis of cancer cells. Although the molecular basis underlying YAP1 subcellular distribution has been extensively explored, it remains to be elucidated how the nuclear localization signal guides YAP1 to pass through the nuclear pore complex. Here, we define a globular type of nuclear localization signal composed of folded WW domains, named as WW-NLS. It directs YAP1 nuclear import through the heterodimeric nuclear transport receptors KPNA-KPNB1, bypassing the canonical nuclear localization signal that has been well documented in KPNA/KPNB1-mediated nuclear import. Strikingly, competitive interference with the function of the WW-NLS significantly attenuates YAP1 nuclear translocation and damages stemness gene activation and sphere formation in malignant breast cancer cells. Our findings elucidate a novel globular type of nuclear localization signal to facilitate nuclear entry of WW-containing proteins including YAP1.

Nuclear dynamics: Formation of bodies and trafficking in plant nuclei.

The existence of the nucleus distinguishes prokaryotes and eukaryotes. Apart from containing most of the genetic material, the nucleus possesses several nuclear bodies composed of protein and RNA molecules. The nucleus is separated from the cytoplasm by a double membrane, regulating the trafficking of molecules in- and outwards. Here, we investigate the composition and function of the different plant nuclear bodies and molecular clues involved in nuclear trafficking. The behavior of the nucleolus, Cajal bodies, dicing bodies, nuclear speckles, cyclophilin-containing bodies, photobodies and DNA damage foci is analyzed in response to different abiotic stresses. Furthermore, we research the literature to collect the different protein localization signals that rule nucleocytoplasmic trafficking. These signals include the different types of nuclear localization signals (NLSs) for nuclear import, and the nuclear export signals (NESs) for nuclear export. In contrast to these unidirectional-movement signals, the existence of nucleocytoplasmic shuttling signals (NSSs) allows bidirectional movement through the nuclear envelope. Likewise, nucleolar signals are also described, which mainly include the nucleolar localization signals (NoLSs) controlling nucleolar import. In contrast, few examples of nucleolar export signals, called nucleoplasmic localization signals (NpLSs) or nucleolar export signals (NoESs), have been reported. The existence of consensus sequences for these localization signals led to the generation of prediction tools, allowing the detection of these signals from an amino acid sequence. Additionally, the effect of high temperatures as well as different post-translational modifications in nuclear and nucleolar import and export is discussed.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

OsWRKY62 and OsWRKY76 Interact with Importin \u03b11s for Negative Regulation of Defensive Responses in Rice Nucleus

Background OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation? Results In this study, we characterized the interaction of OsWRKY62 and OsWRKY76 with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαΔIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαΔIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal (NLS). Similarly, we found that OsIMαΔIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal (NES) in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1-NES fused gene compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae. Conclusion These results revealed the existence of NLS and NES in OsWRKY62. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has been widely used for genome editing. In this system, the cytosine base editor (CBE) and adenine base editor (ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine (C) or adenine (A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE (sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal (NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS (F4NLSr2) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE (sCBE) and F4NLSr2 involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

Faktor Virulensi AvrBs3/PthA pada Ras III, Ras IV, Ras VIII, dan IXO93-068 Patogen Hawar Daun Bakteri (Xanthomonas oryzae pv. oryzae)

<p>AvrBs3/PthA Virulence Factor of Bacterial Leaf Blight<br />Race III, Race IV, Race VIII, and IXO93-068. Dwinita W.<br />Utami, Triny S. Kadir, and Siti Yuriyah. Bacterial leaf<br />blight (BLB) is an important disease of rice and present<br />throughout many of the rice-growing regions in the world,<br />also in Indonesia. Xanthomonas oryzae pv. oryzae (Xoo) is<br />the causal agent and a member of the Protebacteria and like<br />many other this phyllum have a type III secretion system for<br />protein virulence effector (PVE) released on their pathogenicity<br />system. Commonly, PVE in Xanthomonas sp., is<br />coded by AvrBs3/PthA family gene. This research was<br />coducted to identify the virulence factor of AvrBs3/PthA on<br />dominant Indonesian BLB isolates (Race III, Race IV, Ras<br />VIII, and IXO93-068). This objective was obtained by<br />sequence analysis through designed markers for members<br />of the virulence factor AvrBs3/PthA gene family (PthXo4,<br />avrXa7#38, PthXoS and avrXa7sacB50). Results gave information<br />that RaceIII is a dependent elicitor race due to no<br />PVE transcript formed and intraceluler protein target with<br />RLL type on NLS (nuclear localization signal). RaceIV and<br />RaceVIII are the virulent race which PVE active formed with<br />intraceluler protein target and have the RLL and RLLP type<br />for the NLS signal. While isolate IXO93-068 is a virulen<br />isolate that active formed a PVE but the extraceluler protein<br />target is due to no type of NLS. Based on cluster analysis,<br />Race VIII has a genetic distance closely to PthXoS and<br />avrXa7sacB50.</p>

Drosophila proteins involved in metabolism of uracil‐DNA possess different types of nuclear localization signals

Adequate transport of large proteins that function in the nucleus is indispensable for cognate molecular events within this organelle. Selective protein import into the nucleus requires nuclear localization signals (NLS) that are recognized by importin receptors in the cytoplasm. Here we investigated the sequence requirements for nuclear targeting of Drosophila proteins involved in the metabolism of uracil-substituted DNA: the recently identified uracil-DNA degrading factor, dUTPase, and the two uracil-DNA glycosylases present in Drosophila. For the uracil-DNA degrading factor, NLS prediction identified two putative NLS sequences [PEKRKQE(320-326) and PKRKKKR(347-353)]. Truncation and site-directed mutagenesis using YFP reporter constructs showed that only one of these basic stretches is critically required for efficient nuclear localization in insect cells. This segment corresponds to the well-known prototypic NLS of SV40 T-antigen. An almost identical NLS segment is also present in the Drosophila thymine-DNA glycosylase, but no NLS elements were predicted in the single-strand-specific monofunctional uracil-DNA glycosylase homolog protein. This latter protein has a molecular mass of 31 kDa, which may allow NLS-independent transport. For Drosophila dUTPase, two isoforms with distinct features regarding molecular mass and subcellular distribution were recently described. In this study, we characterized the basic PAAKKMKID(10-18) segment of dUTPase, which has been predicted to be a putative NLS by in silico analysis. Deletion studies, using YFP reporter constructs expressed in insect cells, revealed the importance of the PAA(10-12) tripeptide and the ID(17-18) dipeptide, as well as the role of the PAAK(10-13) segment in nuclear localization of dUTPase. We constructed a structural model that shows the molecular basis of such recognition in three dimensions.

A Novel Conserved Nuclear Localization Signal Is Recognized by a Group of Yeast Importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.

Nuclear Translocation Peptides as Antibiotics

Human immunodeficiency virus (HIV) Tat and antimicrobial peptides, such as LL-37, which transfer extraneous DNA to nucleus of host cells are known (5, 6). We performed experiments to find an antimicrobial function for nuclear localization signals (NLS) and nuclear export signals (NES), which are nuclear translocation signals required for shuttling cellular proteins between the nucleus and the cytoplasm (3, 4, 7). Consequently, we obtained evidence that the peptides, which consist of NLS tandems or combinations of NLS and NES, are effective antimicrobial peptides. The antimicrobial activity of these combination peptides for both gram-negative and gram-positive bacteria is very high, and antimicrobial peptides comprised of NLS and NES have the potential to become a new base for peptide antibiotics. The different combinations of NLS and NES within the peptidic design principle comprise eight types of results: the NLS simple sequence, the NLS tandem sequences (NLS+NLS and NLS+NLS+NLS), the NES simple sequence, the NES tandem sequence (NES+NES), the sequence composed of NES linked to an NLS C terminus (NLS+NES), the sequence composed of NLS linked to an NES C terminus (NES+NLS), and the sequence composed of NLS linked to an NLS+NES C terminus (NLS+NES+NLS). The eight types of peptides were synthesized by following the 9-fluorenylmethoxycarbonyl solid-phase synthesis method and purified by reverse-phase high-performance liquid chromatography on a C18 column (purity of ≥95%), and their antimicrobial function against the gram-positive bacterium Staphylococcus aureus IFO12732 and the gram-negative bacterium Escherichia coli IFO12713 was tested using the microbroth dilution method to evaluate their MICs. The results are shown in Table ​Table1.1. The antimicrobial activity for four types of NLS simple sequence (simian virus 40 [SV40] large T antigen, p54, SOX9, and NS5A) was present but weak. However, it was found that antimicrobial function increased remarkably when NLS was made into a tandem sequence (NLS+NLS). More specifically, the antimicrobial performance improved from 30 to 100 times that of NLS on its own. Moreover, the antimicrobial performance results against both gram-positive and gram-negative bacteria further improved several times by making NLS into the three NLS tandem sequence (NLS+NLS+NLS) and the NLS+NES+NLS tandem sequence. Also, NLS-linked NES (NLS+NES, NES+NLS) demonstrated a particularly strong antimicrobial effect against S. aureus. Compared to these results, the NES simple sequence and the NES tandem sequence (NES+NES) did not demonstrate a significant antimicrobial effect. TABLE 1. MICs for combination peptides of NLS and NES To evaluate the peptidic antimicrobial spectrum, MICs for five gram-positive bacterial strains and nine gram-negative bacterial strains were measured for the two types of NLS and NES combination peptides, and the results are shown in Table ​Table2.2. The highly efficient antimicrobial function of the peptide RKKKRKV RKKKRKV, the tandem sequence composed of the modified peptide from SV40 (Table ​(Table2)2) NLS (PKKKRKV), was also confirmed. It is a sequence pair where the Pro in the N terminus was replaced with Arg, showing that some rearrangement could produce better antimicrobial function in NLS/NES combination peptides. TABLE 2. MICs for combination peptides of NLS and NES for various gram-positive and gram-negative bacteria In this study, it was discovered that the antimicrobial function is inherent in the nuclear translocation signal peptide although this peptide does not originally exist for the purpose of self-defense. This is a significant difference from conventional antimicrobial peptides based on biomaterials that exist solely for self-defense (1, 2).

The Requirement of H1 Histones for a Heterodimeric Nuclear Import Receptor

After synthesis in the cytoplasm, H1 histones are imported into the nucleus through an energy-dependent process that can be mediated by an importin beta-importin 7 (Impbeta-Imp7) heterodimer. H1 histones contain two structurally different types of nuclear localization signals (NLS). The first type of NLS resides within the unstructured C-terminal domain and is rich in basic amino acids. In contrast, the highly conserved central domain of the H1 histone contains comparatively few basic amino acids but also represents a functional NLS. The competence for the nuclear import of this globular domain seems to be based on its secondary structure. Here, we show that the Impbeta-Imp7 heterodimer is the only receptor for H1 import. Furthermore, we identified the import receptors mediating the in vitro transport of different NLS of the H1 histone. Using the digitonin-permeabilized cell import assay we show that Impbeta is the most efficient import receptor for the globular domain of H1 histones, whereas the heterodimer of Impbeta and Imp7 is the functional receptor for the entire C-terminal domain. However, short fragments of the C-terminal domain are imported in vitro by at least four different importins, which resembles the import pathway of ribosomal proteins and core histones. In addition, we show that heterodimerization of Impbeta with Imp7 is absolutely necessary for their proper function as an import receptor for H1 histones. These findings point to a chaperone-like function of the heterodimeric complex in addition to its function as an import receptor. It appears that the Impbeta-Imp7 heterodimer is specialized for NLS consisting of extended basic domains.

Using retroviruses to study the nuclear export of mRNA.

Several non-exclusive approaches can be taken to identify and then characterize cellular proteins involved in the nucleocytoplasmic transport of protein or RNA molecules. One approach that has been extremely useful is the genetic identification of factors required for the nucleocytoplasmic transport of a particular macromolecule, e.g. nuclear export of poly(A)+ RNA, by screening yeast conditional mutants for, in this case, nuclear accumulation of poly(A)+ RNA at the restrictive temperature. While this kind of approach has led to the identification of gene products that play a direct or indirect role in several transport pathways, including mRNA export (Amberg et al. 1992; Kadowaki et al. 1994), it can be difficult to subsequently discern the biochemical role played by a particular genetically identified gene product. An alternative strategy is to use protein binding affinity, either in vitro or using the yeast two-hybrid protein:protein interaction assay, to identify factors that specifically bind to a relevant protein target. For example, the G-protein Ran, in its GTP-βound form, is a critical cofactor for nuclear transport mediated by all members of the extensive importin-β/karyopherin-β family of transport factors (Melchior et al. 1993; Moore and Blobel 1993; Izaurralde et al. 1997). The identification of human proteins that can bind to the GTP-, but not GDP-, bound form of Ran in vitro has therefore facilitated the identification of several nuclear transport factors, including CAS and exportin t (Kutay et al. 1997, 1998). Conversely, use of the two-hybrid assay allowed the initial identification of transportin/ karyopherin-β2, the import factor specific for the unusual type of nuclear localization signal (NLS) present in the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1; Pollard et al. 1996; Fridell et al. 1997).

Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase

Bruton's tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. Loss-of-function mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, and the less severe condition xid in mice. Although Btk is mainly localized in the cytoplasm under steady state conditions, it translocates to the plasma membrane upon growth factor stimulation and cross-linking of the B cell receptor. Nevertheless, in ectopically as well as endogenously Btk-expressing cells, it can also translocate to the nucleus. Deletion of the pleckstrin homology (PH) domain (DeltaPH1) leads, however, to an even redistribution of Btk within the nucleus and cytoplasm in the majority of transfected cells. In contrast, an SH3-deleted (DeltaSH3) mutant of Btk has been found to be predominantly nuclear. We also demonstrate that the nuclear accumulation of DeltaPH1 is dependent on Src expression. This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. In addition, while the DeltaPH1 mutant of Btk was found to be active and tyrosine-phosphorylated in vivo, DeltaSH3 displayed decreased autokinase activity and was not phosphorylated. Our findings indicate that the nucleocytoplasmic shuttling of Btk has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B cell development and differentiation.

Deciphering the cellular pathway for transport of poly(A)-binding protein II.

Poly(A)-binding protein II (PABP2) is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 degrees C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.

The nuclear localization signal (NLS) of PDX‐1 is part of the homeodomain and represents a novel type of NLS

The beta-cell homeodomain transcription factor PDX-1 has vital functions both in controlling the expression of pancreatic polypeptide hormones and in the development of the pancreas. The transactivating and DNA-binding properties of PDX-1 have been well characterized, but nuclear transport is still undefined. Here we show that PDX-1 bears a nuclear localization signal (NLS) that is part of helix 3 of the homeodomain. PDX-1 deletion mutants were tagged with enhanced green fluorescent protein (EGFP) and expressed in COS-7 cells. Subcellular localization of the respective PDX-1-EGFP fusion proteins was analyzed by direct fluorescence microscopy and Western immunoblotting using an anti-(GFP). As a result we were able to demonstrate that the homeodomain or helix 3 alone was sufficient and necessary for transport into the nucleus. Point mutations of basic amino acid residues within helix 3 led to identification of an NLS with six amino acids being crucial for nuclear transport of PDX-1. Because this NLS does not match known examples of NLSs, the PDX-1 NLS may represent a novel class of NLS.

Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip.

The sterol regulatory element-binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix-leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin beta. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin beta in the absence of importin alpha. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2-importin beta complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin beta in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix-leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin beta.

Nuclear import and export pathways

Macromolecules enter or leave the nucleus by using nuclear localization signals (NLS), or nuclear export signals (NES), respectively. Different types of NLS and NES are recognized directly or indirectly via adapters, by transport receptors. All transport receptors identified thus far are members of the same family and share an ability to shuttle between the nucleus and the cytoplasm, and to interact with the small GTPase Ran and with nucleoporins at the nuclear pore complex (NPC). The GTPase Ran regulates the interaction of transport receptors with either cargoes, or adapters, or nucleoporins and is crucial in providing directionality to nuclear import and export. Surprisingly, GTP hydrolysis by Ran is not required for translocation of some receptor/cargo complexes through the NPC. One of the challenges for the future will be to establish the mechanisms of translocation through the NPC of different transport receptors together with their cargoes. J. Cell. Biochem. Suppls. 32/33:76-83, 1999.

Nuclear import and export pathways.

Macromolecules enter or leave the nucleus by using nuclear localization signals (NLS), or nuclear export signals (NES), respectively. Different types of NLS and NES are recognized directly or indirectly via adapters, by transport receptors. All transport receptors identified thus far are members of the same family and share an ability to shuttle between the nucleus and the cytoplasm, and to interact with the small GTPase Ran and with nucleoporins at the nuclear pore complex (NPC). The GTPase Ran regulates the interaction of transport receptors with either cargoes, or adapters, or nucleoporins and is crucial in providing directionality to nuclear import and export. Surprisingly, GTP hydrolysis by Ran is not required for translocation of some receptor/cargo complexes through the NPC. One of the challenges for the future will be to establish the mechanisms of translocation through the NPC of different transport receptors together with their cargoes. J. Cell. Biochem. Suppls. 32/33:76-83, 1999.

Nuclear import and export: transport factors, mechanisms and regulation.

Molecules enter and exit the nucleus of eukaryotic cells through aqueous channels formed in the nuclear envelope by nuclear pore complexes (NPC). Proteins entering or leaving the nucleus use nuclear localization signals (NLSs), or nuclear export signals (NESs), respectively. Different types of NLSs and NESs are specifically recognized directly, or indirectly via adapters, by transport receptors. In recent years, many transport receptors, adapters, and the specific cargoes they carry into or out of the nucleus have been identified, revealing an increasing level of complexity. All the transport receptors identified are members of the same family and have in common the ability to shuttle between the nucleus and cytoplasm, and to interact with RanGTP and with nucleoporins at the NPC. The GTPase Ran regulates the interaction between receptors and cargoes, or adapters, and is crucial in providing directionality to nuclear import and export. One of the challenges for the future will be establishing the mechanisms of translocation through the NPC of receptors together with their cargoes. Nuclear import and export of many cargoes is regulated. The molecular mechanisms of regulation of nucleocytoplasmic transport during cell growth, development, viral infections, and different diseases constitute important areas of investigation.

Sequence Determinants for hnRNP I Protein Nuclear Localization

hnRNP I, also referred to as polypyrimidine tract binding protein, is one of the proteins associated with nascent pre-mRNA in the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As for all karyophilic proteins, the nuclear import of hnRNP proteins requires specific sequence determinants that in many instances differ from the canonical import signal. In order to identify the sequences responsible for the nuclear localization, various hnRNP I portions were fused to a reporter protein (bacterial chloramphenicol acetyl transferase) and used in transient transfection assay. By this approach we identified a 60-amino-acid sequence located at the amino terminus of hnRNP I (designated NLD-I) that is both necessary and sufficient for nuclear localization. NLD-I represents a novel bipartite type of nuclear localization signal that bears no resemblance to other nuclear localization determinants so far identified in hnRNP proteins.