RESUMO
The yeast gene BFR1 was originally isolated from a genetic screen for high-copy suppressors of brefeldin A-induced lethality in Saccharomyces cerevisiae. While this result suggested a possible role for the encoded protein, Bfr1p, in the secretory pathway, subsequent data have not fully supported this conclusion. Alternatively, Bfr1p has also been found by yeast two-hybrid analysis to interact with Bbp1p, a component of the spindle pole body. Finally, we have reported that Bfr1p associates with cytoplasmic mRNP complexes containing Scp160p, raising the possibility that Bfr1p may function in mRNA metabolism. Here, we have explored this possibility further. We report that Bfr1p associates with yeast polyribosomes and mRNP complexes even in the absence of Scp160p, and that its interaction with Scp160p-containing mRNP complexes is RNA-dependent. Furthermore, we have determined by fluorescence microscopy and subcellular fractionation that Bfr1p and Scp160p demonstrate similar cytoplasmic localization with enrichment around the nuclear envelope/endoplasmic reticulum. Finally, we report that loss of Bfr1p disrupts the interaction of Scp160p with polyribosomes, thereby demonstrating that the relationship between these two proteins is functional as well as physical. Considered together, these data raise the intriguing possibility that Bfr1p may provide a link between mRNA metabolism, the chromosomal segregation machinery and perhaps secretion in yeast.
Assuntos
Brefeldina A/farmacologia , Resistência Microbiana a Medicamentos , Proteínas Fúngicas/metabolismo , Polirribossomos/metabolismo , RNA Fúngico/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Segregação de Cromossomos , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Polirribossomos/química , Ligação Proteica , Transporte Proteico , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas Repressoras/genética , Ribonucleases/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins. As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein. Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes. Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of> or =1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160 -null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.
Assuntos
Proteínas Fúngicas/química , Proteínas de Membrana/química , Proteínas Nucleares/química , Ribonucleoproteínas/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Sequência de Bases , Primers do DNA/genética , Ácido Edético , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Fúngicos , Substâncias Macromoleculares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peso Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli(A) , Polirribossomos/química , Polirribossomos/metabolismo , Estrutura Terciária de Proteína , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleases , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Ventolin (salbutamol) and Medihaler-Duo (isoprenaline/phenylephrine combination) standard pressurized inhalers were used to administer doses of two or six "puffs" to 16 patients with known reversible airways obstruction. The doses were administered in random order over two days. Both the Ventolin and Medihaler-Duo inhalers substantially increased FEV(1), but in the doses used salbutamol was more effective than isoprenaline/phenylephrine (P < 0.01). There was no significant difference between two and six puffs of salbutamol, though there seemed to be an advantage of six puffs of isoprenaline/phenylephrine over two puffs (P < 0.05). Adrenaline (1/1,000) 0.5 ml and atropine 0.6 mg produced similar increases in FEV(1) to those produced by salbutamol.The Pao(2) fell more than 5 mm Hg in three patients after salbutamol and in three after isoprenaline/phenylephrine. There was no significant fall in mean Pao(2) in any of the treatment groups. It is concluded that the Ventolin inhalant, administered in the conventional dose of two puffs, is as effective a bronchodilator as subcutaneous adrenaline and atropine, is more effective than the Medihaler-Duo, and is without detectable side effects.