Signal-mediated nuclear transport in the amoeba

1999 ◽  
Vol 112 (12) ◽  
pp. 2043-2048 ◽  
Author(s):  
C.M. Feldherr ◽  
D. Akin
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Transport Process ◽  
Nuclear Transport ◽  
Amoeba Proteus ◽  
Regulatory Requirements ◽  
Nuclear Localization Signals ◽  
Evolutionary Changes ◽  
Composition And Structure ◽  
Cytosolic Factors

The evolutionary changes that occur in signal-mediated nuclear transport would be expected to reflect an increasing need to regulate nucleocytoplasmic exchanges as the complexity of organisms increases. This could involve changes in both the composition and structure of the pore complex, as well as the cytosolic factors that mediate transport. In this regard, we investigated the transport process in amoebae (Amoeba proteus and Chaos carolinensis), primitive cells that would be expected to have less stringent regulatory requirements than more complex organisms. Colloidal gold particles, coated with bovine serum albumin (BSA) conjugated with simple (large T) nuclear localization signals (NLSs), bipartite (nucleoplasmin) NLSs or mutant NLSs, were used to assay nuclear import. It was found that in amoebae (1) the diameter of the particles that are able to enter the nucleoplasm is significantly less than in vertebrate cells, (2) the simple NLS is more effective in mediating nuclear import than the bipartite NLS, and (3) the nucleoporins do not appear to be glycosylated. Evidence was also obtained suggesting that, in amoebae, the simple NLS can mediate nuclear export.

A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA

1999 ◽  
Vol 112 (12) ◽  
pp. 2033-2041
Author(s):  
J.J. Ludtke ◽  
G. Zhang ◽  
M.G. Sebestyen ◽  
J.A. Wolff
Keyword(s):  
Active Transport ◽  
Nuclear Localization ◽  
Transport Process ◽  
Nuclear Transport ◽  
Passive Diffusion ◽  
Viral Gene ◽  
Nuclear Localization Signals ◽  
Size Limit ◽  
Cytosolic Extract ◽  
Active Transport Process

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310–1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

The molecular mechanism of translocation through the nuclear pore complex is highly conserved

2002 ◽  
Vol 115 (14) ◽  
pp. 2997-3005
Author(s):  
Carl Feldherr ◽  
Debra Akin ◽  
Trevor Littlewood ◽  
Murray Stewart
Keyword(s):  
Nuclear Import ◽  
Amoeba Proteus ◽  
Nuclear Pore ◽  
Hnrnp A1 ◽  
Transport Mechanisms ◽  
Evolutionary Changes ◽  
Transport Receptors ◽  
Importin Α ◽  
Transport Factors

In this report we investigated the activity of vertebrate nuclear transport factors in a primitive organism, Amoeba proteus, to better understand evolutionary changes in the transport mechanisms of organisms expected to have different requirements for nucleocytoplasmic exchange. It was initially determined that FxFG-containing nucleoporins and Ran, both of which are essential for nuclear import in vertebrates, as well as yeast, are also present and functional in amoebae. This suggests that there are fundamental similarities in the transport process; however, there are also significant differences. Transport substrates containing either the hnRNP A1 M9 shuttling signal (a GST/GFP/M9 fusion protein) or the classical bipartite NLS (colloidal gold coated with BSA-bipartite NLS conjugates), both of which are effectively transported in vertebrate cells, are excluded from the nucleus when microinjected into amoebae. However, when these substrates are injected along with transportin or importin α/β, respectively, the vertebrate receptors for these signals, they readily accumulate in the nucleoplasm. These results indicate that although the molecular recognition of substrates is not well conserved between vertebrates and amoebae, vertebrate transport receptors are functional in A. proteus, showing that the translocation machinery is highly conserved. Since selected nuclear import pathways can be investigated in the absence of competing endogenous transport, A. proteus might provide a useful in vivo system for investigating specific molecular interactions involved in trafficking.

scholarly journals Characterization of Karyopherin Cargoes Reveals Unique Mechanisms of Kap121p-Mediated Nuclear Import

2004 ◽  
Vol 24 (19) ◽  
pp. 8487-8503 ◽  
Author(s):  
Deena M. Leslie ◽  
Wenzhu Zhang ◽  
Benjamin L. Timney ◽  
Brian T. Chait ◽  
Michael P. Rout ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Ribosome Biogenesis ◽  
Nuclear Transport ◽  
Protein Family ◽  
Cellular Functions ◽  
Transport Pathways ◽  
Nuclear Localization Signals ◽  
Insight Into

ABSTRACT In yeast there are at least 14 members of the β-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast β-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.

scholarly journals The Carboxyl Terminus of RNA Helicase A Contains a Bidirectional Nuclear Transport Domain

1999 ◽  
Vol 19 (5) ◽  
pp. 3540-3550 ◽  
Author(s):  
Hengli Tang ◽  
David McDonald ◽  
Tamara Middlesworth ◽  
Thomas J. Hope ◽  
Flossie Wong-Staal
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Rna Helicase ◽  
T Antigen ◽  
Carboxyl Terminus ◽  
Rna Helicase A ◽  
Transport Domain

ABSTRACT Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathioneS-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.

scholarly journals Identification of Nuclear Import and Export Signals within Fli-1: Roles of the Nuclear Import Signals in Fli-1-Dependent Activation of Megakaryocyte-Specific Promoters

2005 ◽  
Vol 25 (8) ◽  
pp. 3087-3108 ◽  
Author(s):  
Wei Hu ◽  
Alana S. Philips ◽  
Juliana C. Kwok ◽  
Michael Eisbacher ◽  
Beng H. Chong
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Nuclear Localization Signals ◽  
Importin Alpha ◽  
Friend Leukemia ◽  
Important Regulator ◽  
Ets Domain

ABSTRACT The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPα) and IMPβ, with NLS1 and NLS2 being predominantly recognized by IMPα and IMPβ, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

1997 ◽  
Vol 75 (5) ◽  
pp. 633-640 ◽  
Author(s):  
Philippe Collas ◽  
Peter Aleström
Keyword(s):  
Nuclear Localization ◽  
Plasmid Dna ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Transport ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
Expression Frequency ◽  
Cytoplasmic Injection

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

scholarly journals Protein-protein docking analysis reveals efficient binding and complex formation between the human nuclear transport proteins

2021 ◽  
Author(s):  
Shravan B Rathod
Keyword(s):  
Binding Energy ◽  
Complex Formation ◽  
Nuclear Export ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Protein Docking ◽  
Transport Proteins ◽  
Stable Complex ◽  
Nuclear Pore ◽  
Nuclear Localization Signals

The nuclear protein transport between the nucleus and cytosol can be considered a core process of cell regulation. Specially designed proteins in nature such as importins, exportins, and some other transporters facilitate this transport in the cell and control the cellular processes. Prior to cargo transports, the transport proteins first recognize the Nuclear localization signals (NLSs) and Nuclear export signals (NESs) of cargo proteins. NLSs and NESs are short peptides that have specific residues which help to locate them on the groove of transport proteins. However, this transport through the Nuclear pore complexes (NPCs) is not possible without the energy supplying protein, RanGTP (RAs-related nuclear protein guanosine-5-triphosphate). In addition to this, transport proteins bind with other similar protein subunits along with RanGTP to transport cargos. Hence, protein-protein binding is a vital step for the cargo movement. Here, I investigated possible bindings between 12 human nuclear transport proteins using protein-protein docking algorithm. Furthermore, the binding energy of docked complexes have been calculated and compared with the experimentally resolved complexes. Among total 78 possible complexes (12 homodimers and 66 Heterodimers), IPO13-IPO13 and KPNA6-TNPO3 were found to have the highest stability. Another complexes such as IPO4-IPO5, IPO4-IPO9, IPO9-IPO13, IPO9-KPNA6, IPO13-KPNA6, and IPO13-XPO5 have the binding energy greater than the -600 kJ/mol which indicates the stable complex formation.

scholarly journals Determination of the Functional Domain Organization of the Importin α Nuclear Import Factor

1998 ◽  
Vol 143 (2) ◽  
pp. 309-318 ◽  
Author(s):  
Andrea Herold ◽  
Ray Truant ◽  
Heather Wiegand ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
T Antigen ◽  
Functional Domain ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
Export Signal

Although importin α (Imp α) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin β nuclear import factor, little is known about the functional domains present in Imp α, with the exception that importin β binding is known to map close to the Imp α NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp α COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp α nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp α nuclear export. Analysis of sequences within Imp α that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp α. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp α may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp α with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

scholarly journals Dimerization of MLH1 and PMS2 Limits Nuclear Localization of MutLα

2003 ◽  
Vol 23 (9) ◽  
pp. 3320-3328 ◽  
Author(s):  
Xiaosheng Wu ◽  
Jeffrey L. Platt ◽  
Marilia Cascalho
Keyword(s):  
Mismatch Repair ◽  
Nuclear Localization ◽  
Dna Sequences ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Genomic Stability ◽  
Dna Damage And Repair ◽  
Nuclear Localization Signals ◽  
Dna Mismatch ◽  
Fine Tune

ABSTRACT DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLα (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLα. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLα may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.

Nuclear transport inEntamoeba histolytica: knowledge gap and therapeutic potential

Parasitology ◽  
2018 ◽  
Vol 145 (11) ◽  
pp. 1378-1387 ◽  
Author(s):  
Marina A. Gwairgi ◽  
Reena Ghildyal
Keyword(s):  
Nuclear Export ◽  
Therapeutic Potential ◽  
Nuclear Transport ◽  
Protozoan Parasite ◽  
Developed Countries ◽  
Transport Mechanisms ◽  
Tropical Regions ◽  
Nuclear Localization Signals ◽  
Invasion Mechanisms ◽  
Cell Components

AbstractEntamoeba histolyticais the protozoan parasite that causes human amoebiasis. It is one of the leading parasitic disease burdens in tropical regions and developing countries, with spread to developed countries through migrants from and travellers to endemic regions.UnderstandingE. histolytica’s invasion mechanisms requires an understanding of how it interacts with external cell components and how it engulfs and kills cells (phagocytosis). Recent research suggests that optimal phagocytosis requires signalling events from the cell surface to the nucleusviathe cytoplasm, and the induction of several factors that are transported to the plasma membrane. Current research in other protozoans suggests the presence of proteins with nuclear localization signals, nuclear export signals and Ran proteins; however, there is limited literature on their functionality and their functional similarity to higher eukaryotes.Based on learnings from the development of antivirals, nuclear transport elements inE. histolyticamay present viable, specific, therapeutic targets.In this review, we aim to summarize our limited knowledge of the eukaryotic nuclear transport mechanisms that are conserved and may function inE. histolytica.

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