| Summary: | 博士 === 國立臺灣大學 === 分子醫學研究所 === 94 === Section I. Functional Characterization of RNA binding protein Rbp1p in Saccharomyces cerevisiae
The Saccharomyces cerevisiae RNA-binding protein Rbp1p was initially identified as a negative growth regulator. Overexpression of Rbp1p can decrease the level of mitochondrial Por1p mRNA by enhancing its degradation. Recently, one kind of cytoplasmic foci had been defined, referred to processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. Here, we show that Rbp1p is found in the mRNA processing bodies. Rbp1p localizes to P-bodies in an xrn1 strain and the N-terminus, C-terminus, and RRM1 domain are involved in this recruitment. We also demonstrate that the N-terminal and RRM1 domains of Rbp1p are necessary but not sufficient for its localization in P bodies. The C-terminus of Rbp1p is involved in oligomers formation. Dhh1p can be localized to P-bodies, and we show that in vivo Rbp1p interacts with Dhh1p. However, this interaction does not affect the recruitment of Rbp1p to P-bodies. Immunostaining also reveal that HA-Rbp1p located to P-bodies in rbp1Δxrn1Δccr4Δ and rbp1Δxrn1Δpan2Δ mutants, suggest that proteins involved in deadenylation are not required for the recruitment of Rbp1p to P-bodies. We also demonstrate that Rbp1p is not required for localizing POR1 mRNA to P bodies in an xrn1Δ strain. We have addressed how the relationship between P-bodies and Rbp1p responds to stress. Our data show that Rbp1p colocalizes with Dhh1p under several stress conditions including glucose deprivation, osmotic stress, and late growth stage. In response to cycloheximide treatment, Dhh1p and Dcp2p are dissociated from P-bodies, but there are no significant differences in Rbp1p localization. Despite the close relationship between Rbp1p and P-bodies, Rbp1p is not essential to P-bodies formation. Based on our data, we propose that Rbp1p is a redundant protein of P-body seed and remains in such foci even when mRNAs are no longer available. In our study, seventeen full-length Rbp1p interacting proteins have identified. Considering that protein-protein interactions play crucial roles in the execution of various biological functions, cumulative connection of these binary interactions would contribute considerably to the functional interpretation of Rbp1p.
Section II. Functional Characterization of Golgi associated protein Vps74p in Saccharomyces cerevisiae
In Saccharomyces cerevisiae, apical bud growth occurs for a brief period in G1 when the deposition of membrane and cell wall is restricted to the tip of the growing bud. Vps74p (product of YDR372C) was recently reported to alter the elongated bud morphology of cdc34-2 cells arrested in the apical growth phase. Here, we show that Vps74p is phosphorylated on serine-19 in a growth-phase-dependent manner. Cdc28 kinase is required for Vps74p phosphorylation. Both alanine and aspartate substitutions in serine-19 directly affect elongated bud morphology of cdc34-2 cells. The alanine substitution abolished Vps74p functional activity on apical growth. In contrast, the aspartate substitution stimulates cellular elongated buds formation. Localization of Vps74p at the Golgi is not dependent on the N-terminal phosphorylation, but on its C-terminal domain. Vps74p is involved in the transportation of GPI-anchored protein Gas1. In addition, deletion of C-terminus, but not N-terminus, of Vps74p affects cell wall integrity. Together, we infer that the distinct functional outcome from Ser-19 phosphorylation modulates Vps74p activity in apical growth, but not in cell wall integrity.
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