Characterization of Posttranslational modification of RNA binding protein, Rbp1p, in Saccharomyces cerevisiae under stress conditions
Date Issued
2009
Date
2009
Author(s)
Tsai, Sung-Pu
Abstract
In eukaryotic cells, RNA binding proteins regulate mRNA transcription and post-transcriptional events, such as splicing, editing, export, stability, localization, and translation. Our laboratory has demonstrated that RNA binding protein1, Rbp1p, appears punctate in cytoplasm and as a negative growth regulator. In addition, Rbp1p localizes to P-bodies and stress granules in cytoplasm upon glucose deprivation and heat shock, respectively. Using two-dimensional electrophoresis analysis, the iso-electric point pattern of Rbp1p shows spots toward acidic pore after glucose deprivation and heat shock treating. The results imply that Rbp1p contains different post-translational modifications in stress conditions. By mass spectrometry analysis, we identify eight phosphorylation sites of Rbp1p (Ser459, Ser462, Ser463, Ser524, Ser526, Thr637, Ser646, and Ser649), and using site-directed mutagenesis approach to substitute alanine for serine/threonine. S646A and S649A mutants of Rbp1p decrease the ability of interaction with Rbp1p by yeast two-hybrid assay. Under glucose deprivation, however, phosphorylation mutants of Rbp1p can still localize to P-bodies, and these mutants cannot affect ability of growth inhibition. In co-immunoprecipitation experiments, I identify that Ssa2p is one of associated proteins with Rbp1p, and the interaction ability diminishes at heat shock. Besides, overexpression of Rbp1p in ssa2Δ strain enhances the growth-inhibition ability. It implies that Ssa2p can inhibit Rbp1p negative regulator growth.
Subjects
Rbp1p
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