Characterization of post-translational modification of RNA binding protein 1, Rbp1p, in Saccharomyces cerevisiae

• Main Authors: Han-Sheng Cheng, 鄭涵聲
• Other Authors: 李芳仁
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/23968879214742655706

碩士 === 國立臺灣大學 === 分子醫學研究所 === 96 === In eukaryotic cells gene expression subjected several level of posttranscriptional regulation included mRNA processing, transport, turnover, and tanslation regulation. Above regulation process involves various of RNA-binding proteins to support. Previously our lab found the Saccharomyces cerevisiae RNA-binding protein Rbp1p was first identified as a negative growth regulator, which contains three copies of an RNA recognition motif (RRM) and two glutamine-rich stretches. We have known that Rbp1p can localize to specific cytoplasm foci called P body when cell growth to stationary phase, glucose deprivation, and osmotic stress. Rbp1p revealed mutiple spot with the same molecular weight but different isoeletric points in 2-DE analysis. This result suggested that Rbp1p contained diverse post-translational modification. In this study using 2-DE analysis found under different growth stage, Rbp1p subjected to different post-translational modification. We speculated that when cell received external stimulus Rbp1p may regulated by post-translational modification. We also found six phosphorylation sites of Rbp1p by means of mass spectrometry. Using site-directed mutagenesis technique to produce phosphorylation sites mutants of Rbp1p. Data revealed simultaneously mutation on serine 459, 462, and 463 to alanine show partial growth inhibition ability lost. Compare to wild Rbp1p the mutant form show different post-translational modification pattern in 2-DE gel. However the mutant of Rbp1p had no effect to localize to P body. In addition we also studied a putative Rbp1p kinase Ksp1p, found that Rbp1p growth inhibition ability lost in Δksp1 strain. Futhermore, in rescue experiment where ADH drove Myc-Ksp1p overexpression in Δksp1 cells, Rbp1p restored its growth inhibition ability. However Ksp1p deletion had no effect on the localization of Rbp1p to P-body. These result suggested thatKsp1p is important for the function of Rbp1p, at least in growth phenotype and post-translational modification. Besides, in this study we determined each of the multiple post-translational modification spots of Rbp1p by mass spectrometry. Moreove we also identified several Rbp1p associated protein included Dhh1p, Hrp1p, Porin1p, Psp1p, and Pub1p.