scholarly journals Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad/Cad)

2000 ◽  
Vol 150 (2) ◽  
pp. 321-334 ◽  
Author(s):  
Delphine Lechardeur ◽  
Luke Drzymala ◽  
Manu Sharma ◽  
Danuta Zylka ◽  
Robert Kinach ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Caspase 3 ◽  
Chromosomal Dna ◽  
Nuclear Targeting ◽  
Mcf7 Cells ◽  
Caspase Activated Dnase ◽  
Localization Signal ◽  
Alternatively Spliced ◽  
Induced Apoptosis ◽  
Spliced Variant

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD–ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3–deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3–dependent regulation of CAD activity takes place.

scholarly journals Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal

PLoS ONE ◽  
2013 ◽  
Vol 8 (11) ◽  
pp. e81387 ◽  
Author(s):  
Rebecca A. Boisvert ◽  
Meghan A. Rego ◽  
Paul A. Azzinaro ◽  
Maurizio Mauro ◽  
Niall G. Howlett
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Targeting ◽  
Localization Signal

scholarly journals Identification of the human c-myc protein nuclear translocation signal.

1988 ◽  
Vol 8 (10) ◽  
pp. 4048-4054 ◽  
Author(s):  
C V Dang ◽  
W M Lee
Keyword(s):  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Vero Cells ◽  
Embryo Cell ◽  
Nuclear Accumulation ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Chicken Muscle ◽  
Localization Signal ◽  
Muscle Pyruvate Kinase

We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.

scholarly journals Essential Role of Nuclear Localization for Yeast Ulp2 SUMO Protease Function

2009 ◽  
Vol 20 (8) ◽  
pp. 2196-2206 ◽  
Author(s):  
Mary B. Kroetz ◽  
Dan Su ◽  
Mark Hochstrasser
Keyword(s):  
Nuclear Localization ◽  
Nuclear Protein ◽  
Catalytic Domain ◽  
Yeast Growth ◽  
Nuclear Targeting ◽  
Sumo Protease ◽  
Sumo Proteases ◽  
Localization Signal

The SUMO protein is covalently attached to many different substrates throughout the cell. This modification is rapidly reversed by SUMO proteases. The Saccharomyces cerevisiae SUMO protease Ulp2 is a nuclear protein required for chromosome stability and cell cycle restart after checkpoint arrest. Ulp2 is related to the human SENP6 protease, also a nuclear protein. All members of the Ulp2/SENP6 family of SUMO proteases have large but poorly conserved N-terminal domains (NTDs) adjacent to the catalytic domain. Ulp2 also has a long C-terminal domain (CTD). We show that CTD deletion has modest effects on yeast growth, but poly-SUMO conjugates accumulate. In contrast, the NTD is essential for Ulp2 function and is required for nuclear targeting. Two short, widely separated sequences within the NTD confer nuclear localization. Efficient Ulp2 import into the nucleus requires the β-importin Kap95, which functions on classical nuclear-localization signal (NLS)-bearing substrates. Remarkably, replacement of the entire >400-residue NTD by a heterologous NLS results in near-normal Ulp2 function. These data demonstrate that nuclear localization of Ulp2 is crucial in vivo, yet only small segments of the NTD provide the key functional elements, explaining the minimal sequence conservation of the NTDs in the Ulp2/SENP6 family of enzymes.

Nuclear Localization Signal(s) Required for Nuclear Targeting of the Maize Regulatory Protein Opaque-2

1992 ◽  
Vol 4 (10) ◽  
pp. 1213
Author(s):  
Marguerite J. Varagona ◽  
Robert J. Schmidt ◽  
Natasha V. Raikhel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Regulatory Protein ◽  
Nuclear Targeting ◽  
Localization Signal

Nuclear targeting of the cauliflower mosaic virus (CaMV) genomeThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 565-571
Author(s):  
Julie Champagne ◽  
Denis Leclerc
Keyword(s):  
Mosaic Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Cell Biology ◽  
Plant Viruses ◽  
Cauliflower Mosaic Virus ◽  
Nuclear Pore ◽  
Regulatory Sequences ◽  
Nuclear Targeting ◽  
Localization Signal

The delivery of the double-stranded DNA viral genome into the nucleus is a critical step for the type member of Caulimoviridae, cauliflower mosaic virus (CaMV). The nucleocapsid (NC) of CaMV is directly involved in this process. A nuclear localization signal located at the N-terminus of the NC was shown to be exposed at the surface of the virion. This nuclear localization signal appears to be important to direct the virus to the nuclear pore complex. The nuclear targeting of the NC needs to be tightly regulated because the process of virus assembly, which also involves the viral NC, occurs in the cytosol. It is now accepted that the N- and C-terminal extensions of the viral NC precursor are efficient regulatory sequences that determine the localization of the viral NC in infected leaves. Proteolytic maturation and phosphorylation of the N- and C-terminal extensions are also important in the regulation of this process. Despite these recent discoveries, the transport of CaMV toward and into the nucleus during early events in the infection cycle remains unclear. In this review, we summarize recent advances that explain the mechanisms of targeting of the CaMV genome to the nucleus and extract from other related animal and plant viruses mechanisms that could hint at the possible strategies used by CaMV to enter the nucleus.

scholarly journals Nuclear Targeting of the Cauliflower Mosaic Virus Coat Protein

1999 ◽  
Vol 73 (1) ◽  
pp. 553-560 ◽  
Author(s):  
Denis Leclerc ◽  
Yvan Chapdelaine ◽  
Thomas Hohn
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Nuclear Localization ◽  
Virus Assembly ◽  
Cauliflower Mosaic Virus ◽  
Nuclear Targeting ◽  
Reading Frame ◽  
Localization Signal ◽  
Viral Coat ◽  
Virus Coat

ABSTRACT The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm.

scholarly journals The Novel Nuclear Targeting and BFRF1-Interacting Domains of BFLF2 Are Essential for Efficient Epstein-Barr Virus Virion Release

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Yu-Ching Dai ◽  
Yen-Tzu Liao ◽  
Yi-Ting Juan ◽  
Yi-Ying Cheng ◽  
Mei-Tzu Su ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
The Novel ◽  
Nuclear Targeting ◽  
Barr Virus ◽  
Nuclear Egress ◽  
Localization Signal ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin β-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids. IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.

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