RESUMO
In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.
Assuntos
Citomegalovirus/genética , Membrana Nuclear/química , Células Vegetais , Proteínas Recombinantes/análise , Proteínas Virais/análise , Expressão Gênica , Células HeLa , Humanos , Imunoprecipitação , Microscopia Confocal , Proteínas Recombinantes/genética , Nicotiana , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genéticaRESUMO
Arabidopsis thaliana contains 18 genes encoding Hsp70s. This heat shock protein superfamily is divided into two sub-families: DnaK and Hsp110/SSE. In order to functionally characterize members of the Hsp70 superfamily, loss-of-function mutants with reduced cytosolic Hsp70 expression were studied. AtHsp70-1 and AtHsp70-2 are constitutively expressed and represent the major cytosolic Hsp70 isoforms under ambient conditions. Analysis of single and double mutants did not reveal any difference compared to wild-type controls. In yeast, SSE protein has been shown to act as a nucleotide exchange factor, essential for Hsp70 function. To test whether members of the Hsp110/SSE sub-family serve essential functions in plants, two members of the sub-family, AtHsp70-14 and AtHsp70-15, were analysed. Both genes are highly homologous and constitutively expressed. Deficiency of AtHsp70-15 but not of AtHsp70-14 led to severe growth retardation. AtHsp70-15-deficient plants were smaller than wild-type and exhibited a slightly different leaf shape. Stomatal closure under ambient conditions and in response to ABA was impaired in the AtHsp70-15 transgenic plants, but ABA-dependent inhibition of germination was not affected. Heat treatment of AtHsp70-15-deficient plants resulted in drastically increased mortality, indicating that AtHsp70-15 plays an essential role during normal growth and in the heat response of Arabidopsis plants. AtHsp70-15-deficient plants are more tolerant to infection by turnip mosaic virus. Comparative transcriptome analysis revealed that AtHsp70-15-deficient plants display a constitutive stress response similar to the cytosolic protein response. Based on these results, AtHsp70-15 is likely to be a key factor in proper folding of cytosolic proteins, and may function as nucleotide exchange factor as proposed for yeast.