The thermolability of nuclear protein import in tsBN2 cells is suppressed by microinjected Ran-GTP or Ran-GDP, but not by RanQ69L or RanT24N

1996 ◽  
Vol 109 (6) ◽  
pp. 1449-1457 ◽  
Author(s):  
A. Dickmanns ◽  
F.R. Bischoff ◽  
C. Marshallsay ◽  
R. Luhrmann ◽  
H. Ponstingl ◽  
...  
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Present Report ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nucleotide Exchange ◽  
Nuclear Protein Import ◽  
Ran Gtp ◽  
Tsbn2 Cells

The nuclear protein regulator of chromosome condensation 1 (RCC1) stimulates guanine nucleotide exchange on a protein, Ran, that is required for nuclear protein import. In the present report, we confirm that RCC1 is also required for nuclear protein import in tsBN2 hamster cells in vivo. The thermolability of nuclear protein import in tsBN2 cells was suppressed by microinjection of purified Ran-GTP into the cytoplasm, but Ran-GDP also relieved the import deficiency, suggesting either that both forms of Ran are active in import in vivo or that tsBN2 cells at restrictive temperature retain a mechanism to convert Ran-GDP to Ran-GTP. To distinguish between these possibilities, nuclear protein import in tsBN2 cells was tested in the presence of Ran mutants, one deficient in GTP hydrolysis (RanQ69L), and one with weak binding to GDP and little or no binding to GTP (RanT24N). Microinjection of the mutant RanQ69L inhibited import in vivo in either the GTP- or GDP-bound form at both the permissive and nonpermissive temperatures. RanT24N-GDP inhibited import in vivo at the permissive temperature and failed to stimulate nuclear protein import at the nonpermissive temperature. The implications of these results for the roles of RCC1 and Ran in nuclear protein import in vivo are discussed.

scholarly journals Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing.

1995 ◽  
Vol 15 (4) ◽  
pp. 2117-2124 ◽  
Author(s):  
M Ren ◽  
A Villamarin ◽  
A Shih ◽  
E Coutavas ◽  
M S Moore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nucleotide Exchange ◽  
Cycle Progression ◽  
Ran Gtpase ◽  
Guanine Nucleotide Exchange ◽  
Nuclear Protein Import ◽  
Ran Gtp

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

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 In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import.

1995 ◽  
Vol 14 (15) ◽  
pp. 3617-3626 ◽  
Author(s):  
N. Imamoto ◽  
T. Shimamoto ◽  
T. Takao ◽  
T. Tachibana ◽  
S. Kose ◽  
...  
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Pore ◽  
Nuclear Protein Import

scholarly journals RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import.

1996 ◽  
Vol 135 (3) ◽  
pp. 559-569 ◽  
Author(s):  
N C Chi ◽  
E J Adam ◽  
G D Visser ◽  
S A Adam
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Gel Filtration ◽  
Nuclear Pore ◽  
Binding Assays ◽  
Permeabilized Cell ◽  
Trimeric Complex ◽  
Cell Extracts ◽  
Nuclear Protein Import ◽  
Ran Gtp

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.

Structural basis for the nuclear protein import cycle

2006 ◽  
Vol 34 (5) ◽  
pp. 701-704 ◽  
Author(s):  
M. Stewart
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Computer Modelling ◽  
Structural Basis ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Importin Α ◽  
Nuclear Protein Import ◽  
Ran Gtp ◽  
Transport Factors

Transport of macromolecules between the nuclear and cytoplasmic compartments through NPCs (nuclear pore complexes) is mediated by soluble transport factors that are commonly members of the importin-β superfamily. In the nuclear protein import cycle, importin-β binds cargo in the cytoplasm (usually via the importin-α adaptor) and transports it through NPCs with which it interacts transiently by way of NPC proteins (‘nucleoporins’) that contain distinctive FG (Phe-Gly) sequence repeats. In the nucleus, Ran-GTP binds to importin-β, dissociating the import complex. The importin-β–Ran-GTP complex recycles to the cytoplasm, whereas importin-α is recycled by the importin-β family member CAS (cellular apoptosis susceptibility protein) complexed with Ran-GTP. Cytoplasmic RanGAP (Ran GTPase-activating protein) dissociates these complexes, freeing the importins for another import cycle. Crystallography and biochemical and cellular studies have enabled a molecular description of the transport cycle to be developed and tested using protein engineering and computer modelling. Importin-β family members are elongated flexible molecules that adapt their shape to encircle their cargoes. Ran-GTP binds at three sites along importin-β and CAS, locking the molecules into a rigid conformation that is unable to bind cargoes effectively. Interactions between transport factors and key nucleoporins (such as Nup1p, Nup2p and Nup50) accelerate the formation and dissolution of many of these complexes.

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 A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

1997 ◽  
Vol 8 (12) ◽  
pp. 2591-2604 ◽  
Author(s):  
Gretchen A. Murphy ◽  
Mary Shannon Moore ◽  
George Drivas ◽  
Pablo Pérez de la Ossa ◽  
Alicia Villamarin ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Direct Role ◽  
Permeabilized Cell ◽  
Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

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.

Sign in / Sign up

Export Citation Format

Share Document