RESUMO
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
Assuntos
Núcleo Celular/genética , Proteínas Fúngicas/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Transporte Biológico , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fusão de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Chaperonas Moleculares , Mutação , Membrana Nuclear/genética , Proteínas Nucleares/metabolismo , Canais de Translocação SEC , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Supressão GenéticaRESUMO
BACKGROUND: Profilins are small eukaryotic proteins involved in modulating the assembly of actin microfilaments in the cytoplasm. They are able to bind both phosphatidylinositol-4,5-bisphosphate and poly-L-proline (PLP) and thus play a critical role in signaling pathways. Plant profilins are of interest because immunological cross-reactivity between pollen and human profilin may be the cause of hay fever and broad allergies to pollens. RESULTS: The determination of the Arabidopsis thaliana profilin isoform I structure, using multiwavelength anomalous diffraction (MAD) to obtain structure-factor phases, is reported here. The structure of Arabidopsis profilin is similar to that of previously determined profilin structures. Conserved amino acid residues in profilins from plants, mammals, and lower eukaryotes are critically important in dictating the geometry of the PLP-binding site and the overall polypeptide fold. The main feature distinguishing plant profilins from other profilins is a solvent-filled pocket located in the most variable region of the fold. CONCLUSIONS: Comparison of the structures of SH3 domains with those of profilins from three distinct sources suggests that the mode of PLP binding may be similar. A comparison of three profilin structures from different families reveals only partial conservation of the actin-binding surface. The proximity of the semi-conserved actin-binding site and the binding pocket characteristic of plant profilins suggests that epitopes encompassing both features are responsible for the cross-reactivity of antibodies between human and plant profilins thought to be responsible for type I allergies.
Assuntos
Arabidopsis/química , Proteínas Contráteis , Proteínas dos Microfilamentos/química , Actinas/química , Actinas/metabolismo , Alérgenos/química , Alérgenos/imunologia , Alérgenos/farmacologia , Sequência de Aminoácidos , Proteínas de Arabidopsis , Sítios de Ligação , Sequência Conservada/genética , Cristalografia por Raios X , Ligação de Hidrogênio , Imunoglobulina E/química , Imunoglobulina E/imunologia , Proteínas dos Microfilamentos/classificação , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Plantas/química , Pólen/imunologia , Pólen/metabolismo , Profilinas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rinite Alérgica Sazonal/metabolismo , Homologia de Sequência de Aminoácidos , Água/metabolismoRESUMO
The bacterial pathogens of the genus Yersinia deliver several virulence factors into target cells using a type III secretion system. We demonstrate that Yersinia protein kinase A (YpkA), an essential bacterial virulence factor, is produced as an inactive serine/threonine kinase. The inactive kinase is activated within the host cell by a cytosolic eukaryotic activator. Using biochemical purification techniques, we demonstrate that actin is a cellular activator of YpkA. This stimulation of YpkA kinase activity by actin depends on the presence of the C-terminal twenty amino acids of YpkA, because deletion of these 20 aa not only obliterates YpkA activity, but it also destroys the interaction between YpkA and actin. Activated YpkA functions within cultured epithelial cells to disrupt the actin cytoskeleton. The disruption of the actin cytoskeleton by YpkA would be expected to inhibit macrophage function and phagocytosis of Yersinia.