scholarly journals Acetylation of nuclear localization signal controls importin-mediated nuclear transport of Ku70

2018 ◽  
Author(s):  
H Fujimoto ◽  
T Ikuta ◽  
A Koike ◽  
M Koike
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Cultured Cells ◽  
Nuclear Transport ◽  
Intracellular Localization ◽  
Lysine Residues ◽  
Importin Α ◽  
Localization Signal ◽  
Transport Factors

AbstractKu70 participates in various intra-and extra-nucleic processes. For multifunctional control, machinery that precisely regulates the intracellular localization of Ku70 is essential. Recently, it was reported that acetylation of Ku70 regulates its function. Here, we demonstrate that specific lysine residues in Ku70 that are targets of acetylation are critical for regulating nuclear transport in vivo. Ku70-GFP fusion proteins transiently expressed in cultured cells localized in the nucleus, whereas mimicking acetylation of K553 or K556 in the Ku70 nuclear localization signal (NLS) by substituting these lysine residues with glutamine markedly decreased the nuclear localization of Ku70. Moreover, the Ku70-importin interaction was suppressed in the K553Q and K556Q mutants. Theoretical estimations indicated that the binding energy between the Ku70 NLS and importin-α decreases with acetylation of lysine residues in the Ku70 NLS, similar to the case when these lysine residues are substituted with glutamine. These results suggest that acetylation of specific lysine residues in the Ku70 NLS is a key switch that controls the localization of Ku70 by modulating interactions between Ku70 and nuclear transport factors.

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 The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells

1995 ◽  
Vol 108 (5) ◽  
pp. 1849-1861 ◽  
Author(s):  
I. Cserpan ◽  
A. Udvardy
Keyword(s):  
Nuclear Localization ◽  
Protein Transport ◽  
Cultured Cells ◽  
Nuclear Transport ◽  
Protein A ◽  
Transport Reaction ◽  
Cytosolic Proteins ◽  
Permeabilized Cells ◽  
Localization Signal

Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction. Phycoerythrine-NLS transport substrate carrying a maximum of 4–5 conjugated NLSs/subunit, or Bsp methyltransferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenously added cytosolic protein. All the characteristic properties of in vivo nuclear transport are faithfully reproduced with these transport substrates: (i) the transport requires a functional NLS in the transported protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear localization peptide efficiently competes for transport, while the transport-incompetent mutant peptide does not; and (iv) wheat germ agglutinin inhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or inefficient extraction of the cytosolic proteins during the permeabilization of the cells. The nuclear transport was proportional to the number of conjugated NLSs. Nuclear transport of phycoerythrine carrying 7–8 conjugated NLSs/subunit required the addition of exogenous cytosolic proteins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport. Nuclear transport with different combinations of transport substrates further supported the assumption that distinct transport mechanisms operate for different substrates. From a mixture of PE-NLS7-8 and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the absence of exogenous cytosol, while Bsp methyltransferase-NLS was efficiently transported. Exogenous cytosol promoted the nuclear transport of the highly conjugated substrate.

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.

Nuclear transport of the U2 snRNP-specific U2B'' protein is mediated by both direct and indirect signalling mechanisms

1994 ◽  
Vol 107 (7) ◽  
pp. 1807-1816 ◽  
Author(s):  
C. Kambach ◽  
I.W. Mattaj
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Transport ◽  
U2 Snrna ◽  
Central Segment ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Localization Signal ◽  
Protein Amino Acids

Experiments investigating the nuclear import of the U2 snRNP-specific B'' protein (U2B'') are presented. U2B'' nuclear transport is shown to be able to occur independently of binding to U2 snRNA. The central segment of the protein (amino acids 90–146) encodes an unusual nuclear localization signal (NLS) that is related to that of the U1 snRNP-specific A protein. However, nuclear import of U2B'' does not depend on this NLS. Sequences in the N-terminal RNP motif of the protein are sufficient to direct nuclear transport, and evidence is presented that the interaction of U2B'' with the U2A' protein mediates this effect. This suggests that U2B'' can ‘piggy-back’ to the nucleus in association with U2A’, and thus be imported to the nucleus by two different mechanisms. U2A' nuclear transport, on the other hand, can occur independently of both U2B'' binding and of U2 snRNA.

A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA

10.1038/11726 ◽  
1999 ◽  
Vol 17 (8) ◽  
pp. 784-787 ◽  
Author(s):  
Lars J. Brandén ◽  
Abdalla J. Mohamed ◽  
C. I. Edvard Smith
Keyword(s):  
Nucleic Acid ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Peptide Nucleic Acid ◽  
Nuclear Transport ◽  
Signal Fusion ◽  
Localization Signal

scholarly journals Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

2011 ◽  
Vol 286 (27) ◽  
pp. 23831-23841 ◽  
Author(s):  
Soma Ghosh ◽  
Alex P. Vassilev ◽  
Junmei Zhang ◽  
Yingming Zhao ◽  
Melvin L. DePamphilis
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Origin Recognition Complex ◽  
Genome Duplication ◽  
Cyclin Dependent Kinase ◽  
Human Origin ◽  
Cell Extracts ◽  
Localization Signal ◽  
Origin Recognition

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2–5) complex in the nucleus, an ORC(1–5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1–6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2–5) are transported independently to the nucleus where they can either assemble into ORC(1–6) or function individually.

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