Aminoacyl-tRNA synthetases (aaRSs)是一群必要的轉譯酵素，每一個胺基酸都有一個相對應的 aaRS，aaRS的主要功能是將胺基酸接到相對應的tRNA上（aa + tRNA — aa-tRNA)，形成的aa-tRNA接 著被延長因子(Elongation Factor)攜帶至核醣體進行蛋白質合成。Arclp是一個酵母菌aaRS的輔助因 子，它不但能結合tRNA，也能與細胞質glutamyl-tRNA synthetase (GluRSc)及 methionyl-tRNA synthetase (MetRS)結合，形成一個三聚體(Arc1p/GluRSc/MetRS)，藉此調控這二個aaRS的催化活性及 細胞內分布。一旦GluRSe及MetRS與Arc1p分離，這二個脫離的aaRS會分別跑去粒線體及細胞核調控 氧化磷酸化相關基因的表達。雖然Arc1p本身不參與竣化(carboxylation)或去竣化(decarboxylation)反 應，也缺乏一般生物素化(biotinylation)蛋白質具有的高度保留序列”AMKM”，它卻是生物素化的自然 標的。這也是酵母菌體內唯一一個不參與CO2轉移反應，卻可被生物素化的蛋白質。Arclp生物素化具 有高度專一性，修飾部位只發生在Arclp蛋白質的K86 (SSKD)，此轉譯後修飾乃由酵母菌內唯一的生 物素化酵素biotin protein ligase (Bpllp)進行催化。然而這個轉譯後修飾對Arclp的生化活性及生物功能 究竟有何影響目前尙不清楚。本計畫爲期三年，我們將深入探討生物素化對Arclp的結構及功能的影 響，並解析生物素化在生物學上的廣泛重要性及應用。具體目標包含：（l)硏究生物素化對Arclp的結 構及功能的影響，（2) Bpllp酵素如何辨認及修飾Arclp。 ;Aminoacyl-tRNA synthetases (aaRSs) are a group of essential translation enzymes, each of which catalyzes the ligation of a specific amino acid to its cognate tRNA (aa + tRNA — aa-tRNA). The resultant aa-tRNAs are then delivered to ribosomes to decipher mRNA codons through base pairing with the anticodon of the aa-tRNA. Arc1p is an aaRS cofactor that binds tRNA and forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the yeast cytoplasm to regulate their catalytic activities and subcellular distributions. Upon dissociation from Arc1p, GluRSc and MetRS are respectively targeted to mitochondria and the nucleus to coordinate OXPHOS (oxidative phosphorylation) gene expression. Despite the fact that Arc1p is not involved in any known carboxylation/decarboxylation reaction and lacks the canonical sequence “AMKM” of biotin-binding domains, it is a natural target of biotin modification. Arc1p is so far the only known yeast protein that is not involved in CO2 transfer, but is biotinylated. Biotinylation of Arc1p is highly specific and occurs only at K86 within the SSKD motif of its N domain. This covalent modification is catalyzed by the one and only biotin protein ligase Bpl1p in yeast. However, the biological significance of this post-translational modification remains elusive. The proposal presented herein elaborates a three-year project, in which we aim to study the effect of biotinylation on Arc1p5s structure and function in particular and the biological significance of this type of post-translational modification in general. Specific aims include (1) elucidating the effect of biotinylation on Arc1p5s structural and function, and (2) delineating the mechanism of Arc1p biotinylation by Bpl1p.
