| Summary: | 博士 === 國立臺灣大學 === 分子醫學研究所 === 91 === Section I. Functional Characterization of Acetyl-CoA Hydrolase, Ach1p, in Saccharomyces cerevisiae
Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By sub-cellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.
Section II. Functional Characterization of RNA-binding protein, Rbp1p, in Saccharomyces cerevisiae
The Saccharomyces cerevisiae RNA-binding protein Rbp1p has been identified as a negative growth regulator; however, its functional role is still obscure. Here, we show that Rbp1p is partly localized to the endoplasmic reticulum-nuclear envelope region and partly to cytoplasmic granules. Recombinant Rbp1p (Rbp1p-dq2 and —dq1,2) lacking one (Rbp1p-dq2) or both (Rbp1-dq1,2) of the glutamine-rich stretches, were localized to mitochondria. Rbp1p, but not those truncated forms (Rbp1p-dN, -dC, -dq1, -dq2 and dq1,2), when overexpressed in yeast, caused defective growth on non-fermentable carbon sources, suggesting that Rbp1p might be involved in mitochondrial function. The level of mitochondrial porin was increased in cells lacking of Rbp1p, whereas overexpression of Rbp1p, but not an N-terminally truncated construct, decreased porin levels. Rbp1p decreased the level of mitochondrial porin mRNA by enhancing its mRNA degradation. Rbp1p interact with porin mRNA 3’-UTR by binding to a G/C(U)n-repeat sequence. These findings demonstrate a role for Rbp1p in the post-transcriptional regulation of mitochondrial porin expression.
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