scholarly journals Structural characterization of human importin alpha 7 in its cargo-free form at 2.5 Å resolution

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
S. Tsimbalyuk ◽  
C. M. Donnelly ◽  
J. K. Forwood
Keyword(s):  
Nuclear Transport ◽  
Free Form ◽  
Nuclear Pore ◽  
Importin Alpha ◽  
Localization Signal ◽  
Health And Disease ◽  
Isoform Specificity ◽  
Alpha 7 ◽  
Structural Insights

AbstractShuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure–function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

scholarly journals Dynamic localization of the nuclear import receptor and its interactions with transport factors.

1996 ◽  
Vol 133 (6) ◽  
pp. 1163-1176 ◽  
Author(s):  
D M Koepp ◽  
D H Wong ◽  
A H Corbett ◽  
P A Silver
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Protein Import ◽  
Genetic Interaction ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Importin Beta ◽  
Importin Alpha ◽  
Localization Signal

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin-beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.

Germline and developmental roles of the nuclear transport factor importin (α)3 in C. elegans

Development ◽  
2001 ◽  
Vol 128 (10) ◽  
pp. 1817-1830 ◽  
Author(s):  
K.G. Geles ◽  
S.A. Adam
Keyword(s):  
Nuclear Transport ◽  
Intracellular Localization ◽  
Nuclear Pore ◽  
P Granules ◽  
C Elegans ◽  
Cellular Events ◽  
Importin Α ◽  
Localization Signal ◽  
Transport Factors ◽  
Embryonic And Larval Development

The importin (α) family of transport factors mediates the nuclear import of classical nuclear localization signal-containing proteins. In order to understand how multiple importin (α) proteins are regulated both in individual cells and in a whole organism, the three importin (α) (ima) genes of Caenorhabditis elegans have been identified and studied. All three IMAs are expressed in the germline; however, only IMA-3 is expressed in the soma. RNA interference (RNAi) experiments demonstrate that IMA-3 is required for the progression of meiotic prophase I during oocyte development. Loss of IMA-3 expression leads also to a disruption of the nuclear pore complex accompanied by the mis-localization of P granules. A range of defects occurring in ima-3(RNAi) F(1) progeny further supports a role for IMA-3 during embryonic and larval development. The functional association of IMA-3 with distinct cellular events, its expression pattern and intracellular localization indicate that regulation of the nuclear transport machinery is involved in the control of developmental pathways.

scholarly journals Interactions between Mad1p and the Nuclear Transport Machinery in the YeastSaccharomyces cerevisiae

2005 ◽  
Vol 16 (9) ◽  
pp. 4362-4374 ◽  
Author(s):  
Robert J. Scott ◽  
C. Patrick Lusk ◽  
David J. Dilworth ◽  
John D. Aitchison ◽  
Richard W. Wozniak
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Nuclear Transport ◽  
Coiled Coil ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Docking Site ◽  
Cellular Functions ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus ◽  
Localization Signal

In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap60p-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the Mlp proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.

scholarly journals Characterization of HIV-1 Vpr Nuclear Import: Analysis of Signals and Pathways

1998 ◽  
Vol 143 (4) ◽  
pp. 875-885 ◽  
Author(s):  
Yonchu Jenkins ◽  
Michele McEntee ◽  
Karsten Weis ◽  
Warner C. Greene
Keyword(s):  
Nuclear Import ◽  
Nuclear Pore ◽  
Nuclear Targeting ◽  
Soluble Receptors ◽  
Preintegration Complex ◽  
Gtp Hydrolysis ◽  
Localization Signal ◽  
Targeting Signals ◽  
Hiv 1

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

scholarly journals Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline

2021 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Timothy Mitchison
Keyword(s):  
Dose Response ◽  
Quantitative Measurement ◽  
Nuclear Transport ◽  
Terminal Region ◽  
Live Cell ◽  
Nuclear Pore ◽  
Antiviral Responses ◽  
Inhibitory Function ◽  
Binding Function

SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest. Using this method, we found that SARS-CoV-2 ORF6 is ~5 times more potent than SARS-CoV-1 ORF6 in inhibiting bidirectional nuclear transport, due to differences in the NUP98-binding C-terminal region that is required for the inhibition. The N-terminal region was also required, but its membrane binding function was dispensable, since loss of the inhibitory function due to N-terminal truncation was rescued by forced oligomerization using a soluble construct. Based on these data, we propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the FG domains of NUP98 at the nuclear pore complex.

Neurochemistry of L-Glutamate Transport in the CNS: A Review of Thirty Years of Progress

2001 ◽  
Vol 66 (9) ◽  
pp. 1315-1340 ◽  
Author(s):  
Vladimir J. Balcar ◽  
Akiko Takamoto ◽  
Yukio Yoneda
Keyword(s):  
Molecular Biology ◽  
Glutamate Transport ◽  
Mammalian Brain ◽  
Activity Data ◽  
System A ◽  
Recent Developments ◽  
The Central Nervous System ◽  
Health And Disease ◽  
Disease Analysis

The review highlights the landmark studies leading from the discovery and initial characterization of the Na+-dependent "high affinity" uptake in the mammalian brain to the cloning of individual transporters and the subsequent expansion of the field into the realm of molecular biology. When the data and hypotheses from 1970's are confronted with the recent developments in the field, we can conclude that the suggestions made nearly thirty years ago were essentially correct: the uptake, mediated by an active transport into neurons and glial cells, serves to control the extracellular concentrations of L-glutamate and prevents the neurotoxicity. The modern techniques of molecular biology may have provided additional data on the nature and location of the transporters but the classical neurochemical approach, using structural analogues of glutamate designed as specific inhibitors or substrates for glutamate transport, has been crucial for the investigations of particular roles that glutamate transport might play in health and disease. Analysis of recent structure/activity data presented in this review has yielded a novel insight into the pharmacological characteristics of L-glutamate transport, suggesting existence of additional heterogeneity in the system, beyond that so far discovered by molecular genetics. More compounds that specifically interact with individual glutamate transporters are urgently needed for more detailed investigations of neurochemical characteristics of glutamatergic transport and its integration into the glutamatergic synapses in the central nervous system. A review with 162 references.

scholarly journals Plasma proteomics of green turtles (Chelonia mydas) reveals pathway shifts and potential biomarker candidates associated with health and disease

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
David P Marancik ◽  
Justin R Perrault ◽  
Lisa M Komoroske ◽  
Jamie A Stoll ◽  
Kristina N Kelley ◽  
...  
Keyword(s):  
Chelonia Mydas ◽  
Sea Turtle ◽  
Protein Abundance ◽  
Plasma Samples ◽  
Important Species ◽  
Green Turtles ◽  
Rehabilitation Programs ◽  
Potential Biomarker ◽  
Health And Disease

Abstract Evaluating sea turtle health can be challenging due to an incomplete understanding of pathophysiologic responses in these species. Proteome characterization of clinical plasma samples can provide insights into disease progression and prospective biomarker targets. A TMT-10-plex-LC–MS/MS platform was used to characterize the plasma proteome of five, juvenile, green turtles (Chelonia mydas) and compare qualitative and quantitative protein changes during moribund and recovered states. The 10 plasma samples yielded a total of 670 unique proteins. Using ≥1.2-fold change in protein abundance as a benchmark for physiologic upregulation or downregulation, 233 (34.8%) were differentially regulated in at least one turtle between moribund and recovered states. Forty-six proteins (6.9%) were differentially regulated in all five turtles with two proteins (0.3%) demonstrating a statistically significant change. A principle component analysis showed protein abundance loosely clustered between moribund samples or recovered samples and for turtles that presented with trauma (n = 3) or as intestinal floaters (n = 2). Gene Ontology terms demonstrated that moribund samples were represented by a higher number of proteins associated with blood coagulation, adaptive immune responses and acute phase response, while recovered turtle samples included a relatively higher number of proteins associated with metabolic processes and response to nutrients. Abundance levels of 48 proteins (7.2%) in moribund samples significantly correlated with total protein, albumin and/or globulin levels quantified by biochemical analysis. Differentially regulated proteins identified with immunologic and physiologic functions are discussed for their possible role in the green turtle pathophysiologic response and for their potential use as diagnostic biomarkers. These findings enhance our ability to interpret sea turtle health and further progress conservation, research and rehabilitation programs for these ecologically important species.

scholarly journals Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

2018 ◽  
Vol 115 (17) ◽  
pp. E3969-E3977 ◽  
Author(s):  
Sasikumar Rajoo ◽  
Pascal Vallotton ◽  
Evgeny Onischenko ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Altered Expression ◽  
Copy Numbers ◽  
Quantitative Image ◽  
Multiple Copies ◽  
Almost All ◽  
High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

scholarly journals SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

1998 ◽  
Vol 140 (3) ◽  
pp. 499-509 ◽  
Author(s):  
Michael J. Matunis ◽  
Jian Wu ◽  
Günter Blobel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Gtpase Activating Protein ◽  
Terminal Domain ◽  
Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

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