RESUMO
RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences ('zipcodes') within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p's C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA-motor protein adapters.
Assuntos
Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Cadeias Pesadas de Miosina , Miosina Tipo V , Miosinas/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Imunofluorescência , Proteínas Fúngicas/genética , Hibridização In Situ , Conformação de Ácido Nucleico , Testes de Precipitina , Ligação Proteica , RNA Fúngico/química , RNA Fúngico/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
By complementation of a salt-sensitive mutant of Saccharomyces cerevisiae, we cloned the SOP1 gene, encoding a 114.5-kDa protein of 1033 amino acids. Cells deleted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress. Following exposure of sop1Delta cells to NaCl stress, the intracellular Na+ level and the Na+/K+ ratio rose to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid identity with Sop1p, produced only slight Na+ sensitivity. Cells carrying a sop1Deltasop2Delta double deletion became, however, hypersensitive to Na+ and exhibited increased sensitivity also to Li+ and K+, suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p revealed a cytoplasmic distribution and cell fractionation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytosolic material. Expression of a Drosophila l(2)gl cDNA in the sop1Deltasop2Delta strain partially restored the Na+ tolerance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor protein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human.
