2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658271摘要：腺甘二磷酸核糖基化因子(ARF)屬於小分子量(~20kDa)的Ras 同源的鳥嘌呤核甘酸結合蛋白家族，包括了ARF 與ARF 近似的蛋白質(ARL)。目前所有測試過的真核細胞都有ARF 與ARL 的存在。ARF 參與細胞內囊狀泡送進、送出等管道的協調訊息工作。ARF的角色可能是經由與Ras 相似的機制作為胞膜上多種蛋白質組合的調節因子，而在真核細胞當中，已發現超過二十種ARL 相關的蛋白質，然而ARL 的功能仍屬未知。我們使用酵母菌來探討酵母菌ARF 和ARL (ARF，ARL)的結構和功能，並發現ARF的功能從人類到酵母菌具有演化上的保留性。酵母菌的兩個ARF 基因yARF1 和yARF2，對於蛋白質從內質網到高基氏體的運送是必需的，而且是酵母菌生存必要的基因。我們曾發現酵母菌的第三個ARF(ARF3)，並證實其功能明顯與前兩個ARF 不同，可能參與酵母菌的出芽生殖和尚未被證實的胞膜傳遞路徑。我們也發現了酵母菌的ARF 近似蛋白質(ARL1，ARL3)，它們和ARFs 有30%-55%的胺基酸序列相似度。ARF 近似蛋白質與ARF 不同的是不能促進Cholera 毒素的腺甘二磷酸核糖基化的能力，也不能活化磷酸脂水解脢D，表示ARL 和ARF 的功能並不相似。ARL1 和ARL3 位於高基氏體，我們將會證實它們經由相似的分子機制，在細胞內胞膜傳遞的過程中扮演重要角色。我們也利用活體實驗以及試管實驗證明了Syt1 為ARL1 的鳥糞嘌呤核甘酸轉換因子 (GEF)，然而ARL1/ARL3 調控細胞內胞膜傳的的分子機制還尚未清楚。我們已經找到一些能調控yARL1 或yARL3 在細胞內結合的分子，將從酵母菌遺傳學、生化學和細胞生物學的角度，繼續探討它們在胞膜傳遞路徑中的功能。我們的最終目標是找出yARL1 和yARL3 參與胞膜傳遞所透過的分子機制，以下是我們目前的研究目標：１. 研究調控Syt1 鳥糞嘌呤核甘酸轉換因子活化ARL1 的分子機制。２. 研究ARL1 在高基氏體上被調控的數條路徑以及分子機制。３. 探討ARL3 的功能及它們的結合分子調控胞膜傳遞的分子機制。這份研究的重要性在於：(1)幫助我們了解ARL1 和ARL3 調控胞膜傳遞的機制。(2)提供關於操作ARL 蛋白質及其相關因子的知識，建立生化學和遺傳學上的實驗操作方法。(3)幫助我們探索因蛋白質運送錯誤而導致的疾病以及與疾病相關的小分子量鳥嘌呤核甘酸結合蛋白，並設計相關的分析與操作策略。<br> Abstract: ADP-ribosylation factor (ARF) and ARF-like (ARL) proteins belong to the ARF family,which regulate several vesicular trafficking pathways as well as cytoskeletal organization.ARF acts in a 'Ras like' manner as a regulator of multi-subuint protein assembly on membranes.ARF function requires cycling between the cytosolic GDP-bound and themembrane-associated, GTP-bound forms. Members of ARF and ARL have been identified inevery eukaryotic cells examined so far. Although more than twenty ARLs have beenidentified in mammalian cells, the biological functions of ARLs remain not understood.I have been using yeast Saccharomyces cerevisiae to investigate the structure and function ofARF and ARL. Two ARF genes (ARF1 and ARF2), found to be essential in the yeast, arerequired for proteins transport from endoplasmic reticulum to the Golgi. We identified thethird yeast ARF gene (ARF3) and demonstrated that the GTPase cycle of Arf3p plays roles inpolarizing growth of the emerging bud and/or an unidentified vesicular trafficking pathway.While investigating the possibility of the existence of other ARF or ARL that may playdifferent roles in vesicular transport, we identified two yeast ARLs (named ARL1, ARL3),which share 30%-55% amino acid identity with the ARFs. We have demonstrated that Arl1pand Arl3p localize to the trans-Golgi network and play important roles in a novel intracellularvesicular transport. We have recently provided both in vivo and in vitro evidence that theyeast Arf-GEF Syt1p is an Arl1p GEF. However, the molecular mechanism ofArl1p/Arl3p-mediated vesicular trafficking is still not clear. We have identified severalmolecules interacted with Arl1p or Arl3p, and will further characterize their functions inArl1p/Arl3p-mediated vesicular trafficking. We will employ yeast genetics, biochemistry, andmolecular cell biology to test this hypothesis. Our ultimate goal is to delineate the cellular andmolecular mechanisms controlling Arl1p and/or Arl3p and their interacting molecules invesicular trafficking.The following studies are our goals in this proposal:1. To investigate molecular mechanism underlying Arl1p activation by Syt1p2. To investigate mechanisms modulating distinct biological activities of Arl1p at the Golgi3. To study roles of Arl3p and its interacting proteins in vesicular traffickingThe proposed research is significant in that: (1) it will contribute to our understanding of theregulation of vesicular trafficking by small GTPases, Arl1p and Arl3p; (2) it will provide theknowledge and the methods for biochemical and genetic manipulation of the interaction ofArl1p and Arl3p and their associated molecules; and (3) the information gained will allow us,in future studies, to discover vesicular trafficking disorder or disease-related small GTPasesand design strategies for analysis and manipulation of these interactions.腺甘二磷酸核糖基化因子(ARF)鳥嘌呤核甘酸結合蛋白鳥糞嘌呤核甘酸轉換因子胞膜傳遞蛋白質運送酵母菌(Saccharomyces cerevisiae)