Dataset Information

The Nucleocapsid Protein of Potato Yellow dwarf Virus: Protein Interactions and Nuclear Import Mediated by a Non-Canonical Nuclear Localization Signal.

ABSTRACT: Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis using fragments of PYDV-N identified a karyophilic region in the carboxy-terminal 122 amino acids. Alanine-scanning mutagenesis was performed across this region in the context of the full-length N protein. Mutants were assayed for their ability to nuclear localize using live-cell imaging and a yeast-based assay. Two amino acid motifs, (419)QKR(421) and (432)KR(433) were shown to be essential for nuclear import and interaction with importin-?. Additional bimolecular fluorescence complementation showed that the PYDV-N-NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or -M. In contrast, interaction with N-NLS mutants appeared to retard the nuclear import of PYDV-P. GFP fused to aa 419-434 established that the PYDV-N-NLS can function outside the context of this protein. Taken together, it was determined that PYDV-N contains the bipartite NLS (419)QKRANEEAPPAAQKR(433).

SUBMITTER: Anderson G

PROVIDER: S-EPMC3355789 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The Nucleocapsid Protein of Potato Yellow dwarf Virus: Protein Interactions and Nuclear Import Mediated by a Non-Canonical Nuclear Localization Signal.

Anderson Gavin G Wang Renyuan R Bandyopadhyay Anindya A Goodin Michael M

Frontiers in plant science 20120202

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis using fr ...[more]

PMID: 22645569

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Project description:Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14-3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14-3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs-HDAC4 and HDAC9.

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Dataset Information

The Nucleocapsid Protein of Potato Yellow dwarf Virus: Protein Interactions and Nuclear Import Mediated by a Non-Canonical Nuclear Localization Signal.

Publications

The Nucleocapsid Protein of Potato Yellow dwarf Virus: Protein Interactions and Nuclear Import Mediated by a Non-Canonical Nuclear Localization Signal.

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