Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

BACKGROUND: The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1-69; however, this targeting sequence has not been experimentally examined or validated. RESULTS: Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin alpha3 and to a very weak extent with importin alpha1 and importin alpha5, the mutant PARP-2 (K36R) did not interact with importin alpha3, providing a molecular explanation why PARP-2 (K36R) is not targeted to the nucleus. CONCLUSION: Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions

Taxonomy of the order Bunyavirales : second update 2018

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).Non peer reviewe

Taxonomy of the family Arenaviridae and the order Bunyavirales : update 2018

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.Peer reviewe

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS-2

With Leptomycin B (LMB) to inhibit nuclear export, followed by immunofluorescence as described in Fig. 2. Representative images are presented.<p><b>Copyright information:</b></p><p>Taken from "Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS"</p><p>http://www.biomedcentral.com/1471-2121/9/39</p><p>BMC Cell Biology 2008;9():39-39.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2496901.</p><p></p

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS-3

Blot using a monoclonal anti-HA antibody. 50 μg of whole cell extracts were used, endogenous PARP-1 levels served as loading control. HEK293T cells were transfected with HA-tagged wild type (wt) PARP-2 or with the PARP-2 mutants K36R and K37R. HA-tagged proteins were detected by immunofluorescence as described for Figure 2A. Representative images are presented.<p><b>Copyright information:</b></p><p>Taken from "Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS"</p><p>http://www.biomedcentral.com/1471-2121/9/39</p><p>BMC Cell Biology 2008;9():39-39.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2496901.</p><p></p

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS-1

Ubsequent detection of HA-tagged proteins by immunofluorescence using an anti-HA antibody and a FITC-conjugated anti-mouse antibody. Representative confocal images are presented. Lysines 19, 20, 36 and 37 of PARP-2 were changed to glutamic acid or methionine as indicated. The nuclear localization is independent of the charge but dependent on the structure of the NLS. As for Fig. 2A, HEK293T cells were transfected with HA-tagged wild type (wt) PARP-2 or with the indicated K → E and K → M mutants and overexpressed proteins were detected as described for Fig. 2A. Representative confocal images are presented.<p><b>Copyright information:</b></p><p>Taken from "Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS"</p><p>http://www.biomedcentral.com/1471-2121/9/39</p><p>BMC Cell Biology 2008;9():39-39.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2496901.</p><p></p

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS-5

Ignments were performed using ClustalW2. Schematic illustration of PARP-2 K → R mutant proteins used in this study: K19, K20, K36 and K37 were changed to arginine using site-directed mutagenesis. Double and quadruple mutants were generated. HA-tagged wild type (wt) PARP-2 or the indicated double or quadruple mutants were expressed in HEK293T cells and expression was analyzed by western blot using a monoclonal anti-HA antibody. 100 μg of whole cell extracts were used, endogenous PARP-1 levels served as loading control.<p><b>Copyright information:</b></p><p>Taken from "Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS"</p><p>http://www.biomedcentral.com/1471-2121/9/39</p><p>BMC Cell Biology 2008;9():39-39.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2496901.</p><p></p

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS-6

Ubsequent detection of HA-tagged proteins by immunofluorescence using an anti-HA antibody and a FITC-conjugated anti-mouse antibody. Representative confocal images are presented. Lysines 19, 20, 36 and 37 of PARP-2 were changed to glutamic acid or methionine as indicated. The nuclear localization is independent of the charge but dependent on the structure of the NLS. As for Fig. 2A, HEK293T cells were transfected with HA-tagged wild type (wt) PARP-2 or with the indicated K → E and K → M mutants and overexpressed proteins were detected as described for Fig. 2A. Representative confocal images are presented.<p><b>Copyright information:</b></p><p>Taken from "Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS"</p><p>http://www.biomedcentral.com/1471-2121/9/39</p><p>BMC Cell Biology 2008;9():39-39.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2496901.</p><p></p

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS-0

Ignments were performed using ClustalW2. Schematic illustration of PARP-2 K → R mutant proteins used in this study: K19, K20, K36 and K37 were changed to arginine using site-directed mutagenesis. Double and quadruple mutants were generated. HA-tagged wild type (wt) PARP-2 or the indicated double or quadruple mutants were expressed in HEK293T cells and expression was analyzed by western blot using a monoclonal anti-HA antibody. 100 μg of whole cell extracts were used, endogenous PARP-1 levels served as loading control.<p><b>Copyright information:</b></p><p>Taken from "Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS"</p><p>http://www.biomedcentral.com/1471-2121/9/39</p><p>BMC Cell Biology 2008;9():39-39.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2496901.</p><p></p