scholarly journals Pol α-primase dependent nuclear localization of the mammalian CST complex

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Joseph M. Kelich ◽  
Harry Papaioannou ◽  
Emmanuel Skordalakes
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Intracellular Localization ◽  
Telomere Maintenance ◽  
Dna Polymerase Alpha ◽  
Localization Signal ◽  
Initiation Of Dna Replication ◽  
Cycle Dependent ◽  
Lagging Strand

AbstractThe human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent. We report an increase in nuclear CST several hours after the initiation of DNA replication, followed by exit from the nucleus prior to mitosis. We identify amino acids of CTC1 involved in Pol α-primase binding and nuclear localization. We conclude, the CST complex does not contain a nuclear localization signal (NLS) and suggest that its nuclear localization is reliant on Pol α-primase. Hypomorphic mutations affecting CST nuclear import are associated with telomere syndromes and cancer, emphasizing the important role of this process in health.

scholarly journals Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit.

1995 ◽  
Vol 15 (2) ◽  
pp. 883-891 ◽  
Author(s):  
M Foiani ◽  
G Liberi ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Posttranslational Modifications ◽  
S Phase ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Initiation Of Dna Replication ◽  
Cycle Dependent

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

scholarly journals The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

1995 ◽  
Vol 130 (2) ◽  
pp. 255-263 ◽  
Author(s):  
T Tagawa ◽  
T Kuroki ◽  
P K Vogt ◽  
K Chida
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Kinase Inhibitors ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Localization Signal ◽  
Cycle Dependent

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

scholarly journals Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

2019 ◽  
Vol 5 (10) ◽  
pp. eaaw7373 ◽  
Author(s):  
Junhua Li ◽  
Jun Zhao ◽  
Simin Xu ◽  
Shu Zhang ◽  
Junjie Zhang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Lytic Replication ◽  
Metabolic Enzyme ◽  
Purine Synthesis ◽  
Localization Signal ◽  
Positively Charged

Protein nuclear translocation is highly regulated and crucial for diverse biological processes. However, our understanding concerning protein nuclear import is incomplete. Here we report that a cellular purine synthesis enzyme inhibits protein nuclear import via deamidation. Employing human Kaposi’s sarcoma-associated herpesvirus (KSHV) to probe the role of protein deamidation, we identified a purine synthesis enzyme, phosphoribosylformylglycinamidine synthetase (PFAS) that inhibits KSHV transcriptional activation. PFAS deamidates the replication transactivator (RTA), a transcription factor crucial for KSHV lytic replication. Mechanistically, deamidation of two asparagines flanking a positively charged nuclear localization signal impaired the binding of RTA to an importin β subunit, thus diminishing RTA nuclear localization and transcriptional activation. Finally, RTA proteins of all gamma herpesviruses appear to be regulated by PFAS-mediated deamidation. These findings uncover an unexpected function of a metabolic enzyme in restricting viral replication and a key role of deamidation in regulating protein nuclear import.

scholarly journals The N-terminus of fission yeast DNA polymerase alpha contains a basic pentapeptide that acts in vivo as a nuclear localization signal.

1995 ◽  
Vol 6 (12) ◽  
pp. 1697-1705 ◽  
Author(s):  
D Bouvier ◽  
G Baldacci
Keyword(s):  
Fission Yeast ◽  
Dna Polymerase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Terminal Sequence ◽  
Nuclear Localization Signals ◽  
Dna Polymerase Alpha ◽  
Localization Signal ◽  
Necessary And Sufficient

The N-terminal sequence of the catalytic subunit of fission yeast DNA polymerase alpha (pol alpha) contains two putative nuclear localization signals (NLS). To check the functionality of these signals in vivo, the N-terminal sequence was experimentally divided into three amino acid blocks, two of which contain a distinct presumptive NLS. Each block was deleted, either individually or in combination with one of the two others. The deleted gene products were expressed in fission yeast, and assayed by indirect immunofluorescence for their aptitude to localize to the cell nucleus. Block II, which contains the putative NLS pentapeptide 97RKRKK, was both necessary and sufficient to promote nuclear import of pol alpha, as well as of a pyruvate kinase fusion protein. Precise excision of the NLS pentapeptide from block II inhibited the nuclear import of pol alpha, thus confirming the role of this sequence as the functional NLS of the fission yeast enzyme.

scholarly journals Two nuclear localization signals regulate intracellular localization of the Duck enteritis virus UL13 protein

2020 ◽  
Author(s):  
Linjiang Yang ◽  
Xixia Hu ◽  
Anchun Cheng ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Intracellular Localization ◽  
Duck Enteritis Virus ◽  
Significant Information ◽  
Nuclear Localization Signals ◽  
Dependent Process ◽  
Importin Α ◽  
Localization Signal ◽  
Enteritis Virus

Abstract Background UL13 multifunctional tegument protein duck enteritis virus (DEV) is predicted as conserved herpesvirus protein kinase (CHPK); however, little is known about its subcellular localization signal. Results In this study, by transfection with two predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein (EGFP), two bipartite nuclear localization signals (NLS) were identified. We found that the NLSs block its nuclear import using ivermectin and proved that nuclear localization signal of DEV UL13 is a classical importin α/β-dependent process. And we constructed the DEV UL13 mutant strain, with the NLSs of DEV UL13 deleted, to explore whether it can affect the virus replication Conclusions The DEV pUL13 amino acids 4 to 7 and 90 to 96 was predicted, and proved that this nuclear import occurs via the classical importin α/β-dependent process. We also found NLSs of pUL13 have no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results would provide significant information for the biological function of pUL13 during DEV infection.

Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits

2001 ◽  
Vol 114 (1) ◽  
pp. 89-99
Author(s):  
J. Bertinato ◽  
C. Schild-Poulter ◽  
R.J. Hache
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Mammalian Cells ◽  
Full Length ◽  
Telomere Maintenance ◽  
Nuclear Localization Signals ◽  
Localization Signal ◽  
Ku Antigen

The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539–556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565–568 containing three lysines was required for the nuclear import of full-length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.

scholarly journals The DNA Polymerase _-Primase Complex: Multiple Functions and Interactions

2003 ◽  
Vol 3 ◽  
pp. 21-33 ◽  
Author(s):  
Marco Muzi-Falconi ◽  
Michele Giannattasio ◽  
Marco Foiani ◽  
Paolo Plevani
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
De Novo ◽  
Telomere Maintenance ◽  
Cellular Processes ◽  
Eukaryotic Dna ◽  
Initiation Of Dna Replication ◽  
Lagging Strand ◽  
Primase Complex

DNA polymerase _ (pol _) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol _ is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA polymerase _-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly.

Nuclear import of glycoconjugates is distinct from the classical NLS pathway

1995 ◽  
Vol 108 (4) ◽  
pp. 1325-1332 ◽  
Author(s):  
E. Duverger ◽  
C. Pellerin-Mendes ◽  
R. Mayer ◽  
A.C. Roche ◽  
M. Monsigny
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Peptide Sequence ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Localization Signal ◽  
Energy Dependent ◽  
Cytosolic Factors

The nuclear import of many proteins depends on a short peptide sequence called the nuclear localization signal. However, glycosylated proteins, which lack such a nuclear localization signal, upon their injection into the cytosol by electroporation, enter the nucleus in a sugar-dependent manner. This paper brings new insights on the mechanism of this process, based on a study of neoglycoprotein nuclear uptake by digitonin-permeabilized cells. The nuclear import of neoglycoproteins is energy dependent: it does not occur when cells are maintained at 4 degrees C or when cells are ATP-depleted by treatment with apyrase. The nuclear import of neoglycoproteins occurs through the nuclear pore: it is inhibited by preincubation of cells with wheat germ agglutinin, a lectin which binds the nuclear pore glycoproteins and blocks the translocation step of nuclear localization signal bearing proteins through the nuclear pore. Furthermore, the nuclear import of neoglycoproteins does not use the pathway of nuclear localization signal bearing proteins: nuclear import of nuclear localization signal bearing proteins depends on cytosolic factors and is inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither requires added cytosolic factors nor is sensitive to alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of nuclear localization signal bearing protein, the nuclear import of neoglycoproteins is not inhibited.

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