真核生物除了細胞質，粒線體也會進行蛋白質的合成，因此在細胞體內必須要有兩套不同的蛋白質合成酵素。ALA1基因是酵母菌(Saccharomyces cerevisiae)中唯一的alanyl-tRNA synthetase (AlaRS)基因，此基因可同時轉譯出兩種AlaRS異構型，分別作用在細胞質及粒線體內。它是利用最靠近5，端的ATG1 轉譯出細胞質的異構型，利用ATG1上游的二個重複ACG (即ACG-25和ACG-24) 作出粒線體異構型。本研究則是著重在進一步探討粒線體AlaRS的細胞內傳輸機制及功能。藉由功能性互補實驗、點突變、及西方式點墨法的測試，我們發現AlaRS的標的訊號不只包括Met1 上游的前序列，尚含括了Met1 下游的18個胺基酸。只有前序列或胺基端18個胺基酸只能將部份AlaRS運送到粒線體內作用，其餘的則殘留在細胞質內。此外，西方點墨法的實驗顯示粒線體AlaRS的基質蛋白質酶切割位似乎不是如PSORTII預測位於Asn16及Thr17之間，而更可能位在上游的序列上。另外在別種低等真核酵母菌: 如C. albicans中我們也發現了其利用唯一的ALA1基因轉譯出細胞質和粒線體的AlaRS蛋白質的例子，同時更確定C. albicans的AlaRS 可以互補於S. cerevisiae AlaRS的生理活性，因此找出其粒線體標的訊號的所在及探討對蛋白質的傳輸影響是接下來重要的課題。總結我們的實驗結果，S. cerevisiae AlaRS的功能除了取決於蛋白質表現量多寡之外，也受到其標的訊號的強弱影響。 In eukaryotic cells, protein synthesis occurs not only in the cytoplasm but also in the mitochondria, so cells need to have more than one set of this enzyme for function in different organelles. It was recently shown that ALA1 is the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase (AlaRS). ALA1 was found to encode both cytoplasmic and mitochondrial isoforms of AlaRS. The former is translationally initiated at the first ATG codon (designated ATG1) at the 5’-end of its open reading frame, while the latter is initiated from upstream in-frame redundant non-ATG codons (i. e., ACG-25 and ACG-24). In this thesis, we focused on the transport mechanism and physiological function of mitochondrial AlaRS. The results of functional tests, site directed mutagenesis, and Western blotting all suggest that the mitochondrial targeting signal of ALA1 includes not only the upstream presequence but also the first eighteen amino acids downstream of Met1 (from codons ACG-25 to TTT+18). When only the presequence or first 18 amino acids of the cytoplasmic N-terminus is used to test for mitochondrial targeting activity, only a portion of the protein products were transported into mitochondria, while the rest remained in the cytoplasm. In addition, the results of Western blotting also reveal that the mitochondrial matrix processing peptidase cutting site may not be between Asn16 and Thr17 as previously predicted by PSORTII, but is rather located at sequences further upstream. In addition, we have also demonstrated that the low eukaryote Candida albicans also uses a single ALA1 gene to encode both cytoplasmic and mitochondrial AlaRS isoforms, and the protein product of this gene has complementing activity in Saccharomyces cerevisiae. Thus finding the mitochondrial targeting signal of this protein and elucidating its effects on protein transport is the next major step of this project. In conclusion, the activity of AlaRS is dependent on both the amount of protein synthesized and the strength of the mitochondrial targeting signal.
