scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

scholarly journals Nuclear Localization Signal and Protein Context both Mediate Importin α Specificity of Nuclear Import Substrates

2006 ◽  
Vol 26 (23) ◽  
pp. 8697-8709 ◽  
Author(s):  
Beate Friedrich ◽  
Christina Quensel ◽  
Thomas Sommer ◽  
Enno Hartmann ◽  
Matthias Köhler
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Import ◽  
Binding Studies ◽  
Vitro Binding ◽  
Importin Α ◽  
Localization Signal ◽  
Experimental Approaches

ABSTRACT The “classical” nuclear protein import pathway depends on importin α and importin β. Importin α binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin β-dependent import pathway. In humans, only one importin β is known to interact with importin α, while six α importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular α importins. Whether the NLS is sufficient to mediate importin α specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin α3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin α binding and transport.

scholarly journals Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

1998 ◽  
Vol 140 (2) ◽  
pp. 259-270 ◽  
Author(s):  
Rohit Mahajan ◽  
Larry Gerace ◽  
Frauke Melchior
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Protein Import ◽  
Nuclear Protein ◽  
Peptide Mapping ◽  
Sufficient Information ◽  
Acceptor Site ◽  
Nuclear Protein Import

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.

scholarly journals Evidence for Distinct Substrate Specificities of Importin α Family Members in Nuclear Protein Import

1999 ◽  
Vol 19 (11) ◽  
pp. 7782-7791 ◽  
Author(s):  
Matthias Köhler ◽  
Christian Speck ◽  
Marret Christiansen ◽  
F. Ralf Bischoff ◽  
Siegfried Prehn ◽  
...  
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Substrate Specificities ◽  
Substrate Preferences ◽  
Importin Α ◽  
Localization Signal ◽  
Nuclear Protein Import ◽  
Human Family ◽  
Import Receptor

ABSTRACT Importin α plays a pivotal role in the classical nuclear protein import pathway. Importin α shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin β-dependent import pathway. In contrast to what is found for importin β, several isoforms of importin α, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin α7. To analyze specific functions of the distinct importin α proteins, we recombinantly expressed and purified five human importin α’s along with importin α from Xenopus andSaccharomyces cerevisiae. Binding affinity studies showed that all importin α proteins from humans or Xenopus bind their import receptor (importin β) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin α proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin α3, whereas most of the other substrates were imported by all importin α proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin α proteins were revealed when two substrates were offered simultaneously.

scholarly journals p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.

1991 ◽  
Vol 115 (5) ◽  
pp. 1203-1212 ◽  
Author(s):  
D A Jans ◽  
M J Ackermann ◽  
J R Bischoff ◽  
D H Beach ◽  
R Peters
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
T Antigen ◽  
Regulatory Proteins ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Kinetic Measurements ◽  
Nuclear Protein Import

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.

scholarly journals A GTPase distinct from Ran is involved in nuclear protein import.

1996 ◽  
Vol 133 (5) ◽  
pp. 971-983 ◽  
Author(s):  
D J Sweet ◽  
L Gerace
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Inhibitory Effect ◽  
Nuclear Pore ◽  
Mutant Form ◽  
Direct Role ◽  
Nuclear Protein Import ◽  
Dependent Transport

Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.

scholarly journals Nuclear Import of Adenovirus DNAin VitroInvolves the Nuclear Protein Import Pathway and hsc70

2000 ◽  
Vol 275 (6) ◽  
pp. 4298-4304 ◽  
Author(s):  
Andrew C. S. Saphire ◽  
Tinglu Guan ◽  
Eric C. Schirmer ◽  
Glen R. Nemerow ◽  
Larry Gerace
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Protein Import

Importin α/β-mediated nuclear protein import is regulated in a cell cycle-dependent manner

2004 ◽  
Vol 297 (1) ◽  
pp. 285-293 ◽  
Author(s):  
Noriko Yasuhara ◽  
Eri Takeda ◽  
Hitomi Inoue ◽  
Ippei Kotera ◽  
Yoshihiro Yoneda
Keyword(s):  
Cell Cycle ◽  
Protein Import ◽  
Nuclear Protein ◽  
Dependent Manner ◽  
Importin Α ◽  
A Cell ◽  
Nuclear Protein Import ◽  
Cycle Dependent

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

scholarly journals The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

2002 ◽  
Vol 158 (1) ◽  
pp. 63-77 ◽  
Author(s):  
Tobias C. Walther ◽  
Helen S. Pickersgill ◽  
Volker C. Cordes ◽  
Martin W. Goldberg ◽  
Terry D. Allen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Slight Reduction ◽  
Cytoplasmic Filaments ◽  
Localization Sequence ◽  
Importin Α

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.

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