Identificação e caracterização de alvos celulares da proteína NIG (NSP-Interacting GTPase)

• Autor(a) principal: Machado, João Paulo Batista
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2439
• topic: (NSP-Interacting GTPase); Alvos celulares; (NSP-Interacting GTPase); Cellular targets; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

The geminivirus nuclear shuttle protein (NSP) facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts along with the movement protein (MP) to translocate the viral DNA to adjacent cells. However, the mechanism by which NSP mediates the nucleocytoplasmic movement of the viral DNA is unknown. Recently, a GTPase, designated NIG (NSP-interacting GTPase), which displays biochemical and structural properties consistent with a role in the nucleocytoplasmic transport of viral DNA, has been identified. NIG may act as a cellular cofactor for NSP function. To assess the potential role of NIG in general cellular nucleocytoplasmic transport of protein complexes, we performed yeast two-hybrid screens with a Pro-rich domain of NIG as bait. The yeast strain AH109, previously transformed with pBD-Pro-Rich, was cotransformed with a cDNA library from Arabidopsis thaliana cloned into pEXPAD502 vector. Double transformants were selected on medium lacking leucine and tryptophan. To select for interactions between the bait BD-Pro-Rich and Arabidopsis cDNA library-encoded proteins, double transformants were plated on medium lacking His and supplemented with 10mM 3AT. Among five isolated cDNAs that displayed His prototrophy and β-galactosidase activity, CSN5A and At2G41020 were selected for further analyses. CSN5A (COP9 Signalosome 5A) is one of the components of COP9 signalosome complex (CSN) and corresponds to the subunit responsible for the isopeptidase activity displayed by this complex. The function of the deduced protein from At2G41020 is unknown but has been associated with the spliceosome machinery due to its homology to the human Npw38. Interactions between NIG and CSN5A or At2G41020 were also detected in vivo by bimolecular fluorescence complementation (BiFC) assays and appear to occur in the nucleus and in cytosolic aggregates. The in vivo interaction between NIG and CSN5A was further confirmed by co-immunoprecipitation assays. A CSN5A-GFP fusion was located in the nucleus and cytoplasm, whereas At2G41020-GFP was predominantly localized in the nucleus, consistent with its possible involvement with the spliceosome. Although NIG has been shown to be located in the cytoplasm, co-expression of YFP-NIG with CSN5AGFP or At2G41020-GFP resulted in its relocation to the nucleus. Collectively, these results indicate that CSN5A and At2G41020 may be targets for the elucidation of the functional role of NIG in the cellular transport of macromolecules.