中文摘要 在Saccharomyces cerevisiae 中，細胞質及粒線體功能的alanyl-tRNA synthetase (簡寫為AlaRS) 是由ALA1 基因所提供。ALA1 基因利用最靠近 mRNA 5’ 端的AUG1 轉譯出細胞質的異構型，利用AUG1 上游的二個重複 ACG (即ACG-25 和ACG-24) 作出粒線體異構型。在本實驗中我們選殖了 Candida albicans 唯一負責alanyl-tRNA synthetase 的基因 (即CaALA1) ，經 由物種間的功能性基因互補測試，我們發現CaALA1 可以取代S. cerevisiae ALA1 的細胞質及粒線體功能。然而，不同於S. cerevisiae 的轉譯起始機制， CaALA1 是利用第一個AUG (即AUG1) 為起始密碼，作出一個同時具有粒線 體及細胞質功能的蛋白質，當AUG1 點突變後，由於失去蛋白質的起始作用， 因此失去粒線體蛋白質的功能，但是CaALA1 可以利用第二個AUG (即AUG9) 為起始密碼，作出細胞質的蛋白質。接著我們利用細胞質功能的valyl-tRNA synthetase 為回報基因進一步找出其粒線體標的訊號的範圍為包含N端第1 ~ 42 個胺基酸，此外AUG1 所做出的前導序列 (胺基酸1 ~ 42) 可以有效地將 回報基因帶到粒線體中。 在本論文的第二部分研究則是著重在進一步探討粒線體ScAlaRS 的細 胞內傳輸機制及功能。藉由功能性互補試驗、點突變、及西方氏點墨法的測 試，我們發現AlaRS 的標的訊號不只包括Met1 上游的前導序列 (胺基酸-25 ~ -1)，尚涵括了Met1 下游的18 個胺基酸。只有前導序列或胺基端18 個胺 基酸只能將小部份ScAlaRS 運送到粒線體內作用。從ACG-25 起始的蛋白質 會全部運送到粒線體中作用；反之從AUG1 起始的蛋白質幾乎全部都存在於 細胞質中。值得注意的是，AlaRS 的功能除了取決於其標的訊號的長度，也 受到其蛋白質表現量多寡的影響。 It was previously shown that the cytoplasmic and mitochondrial activities of alanyl-tRNA synthetase of Saccharomyces cerevisiae are provided by two distinct translational products of ALA1, one initiated at the AUG codon closest to the 5’-end of its mRNA transcripts and the other at upstream in-frame redundant non-AUG codons (i.e., ACG-25 and ACG-24). We report here the cloning and characterization of the only alanyl-tRNA synthetase gene of Candida albicans (designated as CaALA1). Cross-species complementation assays suggest that CaALA1 can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae. However, unlike the case in S. cerevisiae, both of these two activities are provided a single primary translational product of CaALA1 that is initiated from the first AUG codon (designated as AUG1) on its coding sequence. When the first AUG codon is inactivated by point mutations, the mitochondrial function of this gene is impaired, presumably due to inability to initiate protein synthesis, while its cytoplasmic function can be rescued by alternative initiation at a downstream AUG codon, AUG9. Functional mapping using the cytoplasmic form of valyl-tRNA synthetase as the passenger protein identifies the first 42 amino-terminal residues of the Candida protein as the mitochondrial targeting signal. In addition, evidence shows that an exclusive mitochondrial targeting signal is translationally initiated from the first AUG initiator. In the second part of this thesis, we focus on the transport mechanism and physiological function of mitochondrial ScAlaRS. The results of functional tests, site-directed mutagenesis, and western blotting all suggest that the mitochondrial targeting signal of ScAlaRS includes not only the leader peptide but also the first eighteen amino acids downstream of Met1. Neither the leader peptide nor the eighteen-residue peptide can function as a transit signal per se. By western blotting assays, our data shows that the ACG-25-initiated proteins are fully translocated into the mitochondria, and the AUG1-initiated proteins are almost completely retained in the cytosol. These results confirm our previous observations that cytoplasmic and mitochondrial activities of ScALA1 are provided by two distinct translational products. In addition, partition of ScAlaRS is determined by both its leader peptide and expression levels.
