scholarly journals Defective nuclear import of Tpr in Progeria reflects the Ran sensitivity of large cargo transport

2013 ◽  
Vol 201 (4) ◽  
pp. 541-557 ◽  
Author(s):  
Chelsi J. Snow ◽  
Ashraf Dar ◽  
Anindya Dutta ◽  
Ralph H. Kehlenbach ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Import ◽  
Protein Complexes ◽  
Nucleocytoplasmic Transport ◽  
Large Protein ◽  
Localization Signal ◽  
Import Receptors ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome ◽  
Major Defect ◽  
Import Receptor

The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)–containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.

scholarly journals Transportin-SR, a Nuclear Import Receptor for SR Proteins

1999 ◽  
Vol 145 (6) ◽  
pp. 1145-1152 ◽  
Author(s):  
Naoyuki Kataoka ◽  
Jennifer L. Bachorik ◽  
Gideon Dreyfuss
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Sequence Similarity ◽  
Sr Proteins ◽  
Amino Acid Sequence Similarity ◽  
Sr Protein ◽  
Localization Signal ◽  
Import Receptors ◽  
Import Receptor

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain–bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain– bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin β/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.

scholarly journals A pathway linking oxidative stress and the Ran GTPase system in progeria

2014 ◽  
Vol 25 (8) ◽  
pp. 1202-1215 ◽  
Author(s):  
Sutirtha Datta ◽  
Chelsi J. Snow ◽  
Bryce M. Paschal
Keyword(s):  
Oxidative Stress ◽  
Nuclear Membrane ◽  
Nucleocytoplasmic Transport ◽  
Lamin A ◽  
Mutant Form ◽  
Ran Gtpase ◽  
Nuclear Levels ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome ◽  
The Relationship

Maintaining the Ran GTPase at a proper concentration in the nucleus is important for nucleocytoplasmic transport. Previously we found that nuclear levels of Ran are reduced in cells from patients with Hutchinson–Gilford progeria syndrome (HGPS), a disease caused by constitutive attachment of a mutant form of lamin A (termed progerin) to the nuclear membrane. Here we explore the relationship between progerin, the Ran GTPase, and oxidative stress. Stable attachment of progerin to the nuclear membrane disrupts the Ran gradient and results in cytoplasmic localization of Ubc9, a Ran-dependent import cargo. Ran and Ubc9 disruption can be induced reversibly with H2O2. CHO cells preadapted to oxidative stress resist the effects of progerin on Ran and Ubc9. Given that HGPS-patient fibroblasts display elevated ROS, these data suggest that progerin inhibits nuclear transport via oxidative stress. A drug that inhibits pre–lamin A cleavage mimics the effects of progerin by disrupting the Ran gradient, but the effects on Ran are observed before a substantial ROS increase. Moreover, reducing the nuclear concentration of Ran is sufficient to induce ROS irrespective of progerin. We speculate that oxidative stress caused by progerin may occur upstream or downstream of Ran, depending on the cell type and physiological setting.

scholarly journals Nuclear Import Receptors and hnRNP K Mediates Nuclear and Stress Granule Localization of SIRLOIN

2021 ◽  
Author(s):  
Jialin Yao ◽  
Qiao Zhou ◽  
Hengyi Xiao ◽  
Da Jia ◽  
Qingxiang Sun
Keyword(s):  
Nuclear Import ◽  
Rna Binding ◽  
Weak Interactions ◽  
Underlying Mechanism ◽  
Stress Granule ◽  
Hnrnp K ◽  
Permeabilized Cells ◽  
Import Receptors ◽  
Rna Motif ◽  
Import Receptor

Abstract The majority of lncRNAs and a small fraction of mRNAs localize in the cell nucleus to exert their functions. A SIRLOIN RNA motif was previously reported to drive its nuclear localization by the RNA-binding protein hnRNP K. However, the underlying mechanism remains unclear. Here, we report crystal structures of hnRNP K in complex with SIRLOIN, and with the nuclear import receptor (NIR) Impα1, respectively. The protein hnRNP K bound to SIRLOIN with multiple weak interactions, and interacted Impα1 using an independent high-affinity site. Forming a complex with hnRNP K and Impα1 was essential for the nuclear and stress granule localization of SIRLOIN in semi-permeabilized cells. Nuclear import of SIRLOIN enhanced with increasing NIR concentrations, but its stress granule localization peaked at a low NIR concentration. Collectively, we propose a mechanism of SIRLOIN localization, in which NIRs functioned as drivers/regulators, and hnRNP K as an adaptor.

scholarly journals A Conserved DrosophilaTransportin-Serine/Arginine-rich (SR) Protein Permits Nuclear Import ofDrosophila SR Protein Splicing Factors and Their Antagonist Repressor Splicing Factor 1

2002 ◽  
Vol 13 (7) ◽  
pp. 2436-2447 ◽  
Author(s):  
Eric Allemand ◽  
Svetlana Dokudovskaya ◽  
Rémy Bordonné ◽  
Jamal Tazi
Keyword(s):  
Nuclear Import ◽  
Direct Interaction ◽  
Sr Proteins ◽  
Splicing Factor ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Nuclear Factors ◽  
Localization Signal ◽  
Import Receptors ◽  
Splicing Factor 1

Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by whichDrosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophilaimportin β-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.

scholarly journals Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

2004 ◽  
Vol 15 (5) ◽  
pp. 2276-2286 ◽  
Author(s):  
Neal D. Freedman ◽  
Keith R. Yamamoto
Keyword(s):  
Glucocorticoid Receptor ◽  
Nuclear Import ◽  
Hormonal Control ◽  
Nuclear Localization Signals ◽  
Cell Extracts ◽  
Importin Α ◽  
Import Receptors ◽  
Unidentified Component ◽  
Import Receptor

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin α selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin α-importin β heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin α bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.

scholarly journals The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
John Frederick Beckmann ◽  
Gagan Deep Sharma ◽  
Luis Mendez ◽  
Hongli Chen ◽  
Mark Hochstrasser
Keyword(s):  
Protein Interactions ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Cytoplasmic Incompatibility ◽  
Host Protein ◽  
Localization Signal ◽  
Histone Exchange ◽  
Embryonic Viability ◽  
Import Receptor

Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where we identified both host effects and high-copy suppressors of toxicity. The strongest suppressor was karyopherin-α, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-α; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-α, P32, or CidA in female flies suppressed CI. We propose that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.

scholarly journals The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1650
Author(s):  
Angela R. Harrison ◽  
Cassandra T. David ◽  
Stephen M. Rawlinson ◽  
Gregory W. Moseley
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Ebola Virus ◽  
Molecular Mapping ◽  
Nucleocytoplasmic Transport ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Trafficking ◽  
C Terminus ◽  
Import Receptors

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

scholarly journals An epilepsy-associated mutation in the nuclear import receptor KPNA7 reduces nuclear localization signal binding

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Luke T. Oostdyk ◽  
Zhenjia Wang ◽  
Chongzhi Zang ◽  
Hui Li ◽  
Michael J. McConnell ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Localization Signal ◽  
Import Receptor

scholarly journals Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1

2020 ◽  
Vol 21 (7) ◽  
pp. 2650
Author(s):  
Kamalakannan Radhakrishnan ◽  
Seon-Joo Park ◽  
Seok Won Kim ◽  
Gurusamy Hariharasudhan ◽  
Seo-Yeon Jeong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Ring Finger ◽  
Nucleocytoplasmic Transport ◽  
Ring Finger Protein ◽  
Finger Protein ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989–1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

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