色質體的蛋白質分送與運輸

Abstract

第一章-能將外源蛋白高效率送往白色體的運輸胜肽 色質體（plastids）為植物特有且必需的胞器，在不同組織分化成不同功能的胞器，例如葉綠體（chloroplasts）、雜色體（chromoplasts）與白色體（leucoplasts）等。白色體存在於非光合作用組織，如白色的根、塊莖、種子和花瓣等，它們負責許多養分的製造與儲存。色質體蛋白是由核基因轉錄，在細胞質轉譯產生前驅蛋白（precursors）後，接著透過N端的運輸胜肽（transit peptide）將蛋白送往色質體執行其蛋白功能。目前已知，不同運輸胜肽具有不同色質體的偏好性。大多數的運輸胜肽選殖於葉綠體的蛋白質，如RBCS運輸胜肽（RBCStp），但對於能將蛋白送往白色體的運輸胜肽知道的很少。根據先前實驗室成員利用色質體蛋白離體運送（in vitro plastid protein import）實驗，選殖數個具高效率白色體運送的運輸胜肽，接上綠色螢光蛋白（green fluorescence protein, GFP），進行阿拉伯芥（Arabidopsis）轉殖，常用的RBCStp也接上GFP並進行Arabidopsis轉殖，作為比較。我從轉殖植物中，分離到含運輸胜肽-GFP轉殖基因同型合子的T4子代，萃取其根部及葉片的RNA與蛋白質，進行GFP運送到白色體效率的分析與比較，發現這些運輸胜肽將GFP送往白色體的效率比RBCStp高出四倍以上，同時大部分的運輸胜肽GFP送往葉綠體的效率也較RBCStp好一些。另外，我也以顯微鏡方法觀察GFP在根部及花瓣的表現，結果顯示不同運輸胜肽對花瓣與根的白色體有不同的偏好性。總合而言，這些運輸胜肽對於未來改良植物白色體蛋白或於白色體大量表現外源蛋白將有很大助益。 第二章-尋找協助蛋白質運送至胞器之分送因子 粒線體與葉綠體是植物重要的胞器，分別負責產生ATP能量與光合作用。而這些胞器的蛋白質大多都由細胞核所編碼，在細胞質轉譯為前驅蛋白質。而這些前驅蛋白質N端帶有運輸訊號（targeting signal），通過胞器膜上的運轉機組（translocon complex）進入到胞器中。至於前驅蛋白質是如何被準確地從細胞質中送往粒線體或葉綠體的表面，其機制尚未清楚。儘管某些伴隨蛋白（chaperone），已推測與前驅蛋白的分送有關，但這些伴隨蛋白不具專一性，因此對於特定將粒線體與葉綠體前驅蛋白運送的分送因子（sorting factor）目前尚未了解。於是為了找尋這些因子，我們設計了一套遺傳篩選方法，將葉綠體芳香族胺基酸生合成路徑（shikimic acid pathway）中的EPSP生產酵素（EPSPS），改變其胺基酸序列（EPSPS-TIPS），讓其可抗除草劑glyphosate，接著將此酵素N端與粒線體蛋白AOX（alternative oxidase）的運輸訊號融合並轉殖到阿拉伯芥中。再利用12.5 μM濃度的除草劑glyphosate篩選12天。若分送因子被突變，會使得前驅蛋白無法被順利送往粒線體，有可能改而送到葉綠體，則此突變株因EPSPS-TIPS進入葉綠體而抗glyphosate，我以能夠在12.5 μM glyphosate上生長出真葉的植株視為抗性植株，接著透過PCR mapping與次世代定序的方法找尋突變位置，希望能因此尋找到協助前驅蛋白正確運送至特定胞器的分送因子。

Chapter 1 - High efficiency transit peptides for protein transport into leucoplasts Plastids differentiate into various functional types in different tissues, for instance, chloroplasts in leaves for photosynthesis, chromoplasts in orange-colored fruits for carotenoid synthesis, and leucoplasts for nutrient synthesis and storage in white-colored roots and seeds. Plastid proteins are encoded by the nucleus and are translated in the cytosol. Under the direction of the transit peptide at the N-terminus, the proteins are targeted to plastids to function. It is known that different transit peptides prefer different plastid types. Most transit peptides characterized so far are for targeting proteins to chloroplasts. There is no tool available for high efficiency delivery of proteins into leucoplasts. However, leucoplasts are the plastids in major human food crops like rice, wheat, and potatoes. We therefore selected a few transit peptides that show high import efficiency into leucoplasts in vitro. We transformed GFP fusion constructs of these transit peptides into Arabidopsis thaliana. GFP fusion with the transit peptide of the precursor to the small subunit of RuBP carboxylase (RBCStp), the most commonly used chloroplast-targeting transit peptide, was also constructed and transformed into Arabidopsis to be used as a control. Then I used real-time PCR, western blotting and microscopy to analyze the efficiency of these transit peptides in delivering GFP proteins to leucoplasts. My results show that our candidate transit peptides all have higher efficiency in delivering GFP to leucoplasts than RBCStp. Interestingly, most candidate transit peptides also have better efficiency in delivering GFP to leaf chloroplasts than RBCStp. Meanwhile, I found that different transit peptides show different preference between petals and roots. In summary, these transit peptides could be valuable tools for delivering desired proteins into leucoplasts for biotechnology applications in the future. Chapter 2 - Genetic screening to identify cytosolic sorting factors for chloroplast and mitochondrion protein import In plant cells, mitochondria and chloroplast are essential organelles that perform important functions such as ATP production and photosynthesis. Most proteins in mitochondria and chloroplasts are encoded by the nuclear genome. These proteins are first synthesized in the cytosol as precursor proteins with an N-terminal targeting signal. How precursor proteins are targeted from the cytosol to the mitochondrion or chloroplast is unknown. Although some general chaperone proteins, such as HSP70 and 14-3-3 proteins, have been shown to interact with cytosolic precursor proteins, no factors that can specifically target cytosolic precursor proteins to chloroplasts or mitochondria have been identified. To identify cytosolic targeting factors for mitochondria and chloroplasts, we designed a genetic screen for identifying Arabidopsis mutants defective in protein sorting to mitochondria. We fused the mitochondrial targeting signal of alternative oxidase (AOX) to the N terminus of an EPSP synthase variant (EPSPS-TIPS), which confers glyphosate resistance when localized in chloroplasts. We transformed the fusion construct into wild-type Arabidopsis. Mutagenized M2 seeds of the transformant were grown on MS medium containing 12.5 μM glyphosate for 12 days. Plants with expanded true leaves, which suggest potential glyphosate resistance, were selected. The mutant locus of a confirmed glyphosate resistant mutant was mapped by PCR-based markers and next generation sequencing. The mutation was mapped to the gene AT2G28800, which is named ALB3 (albino 3) because loss-of-function mutations of this gene cause an albino phenotype.