李芳仁臺灣大學：分子醫學研究所張逸蘋Chang, Yi-PinYi-PinChang2007-11-262018-07-092007-11-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/51374腺核苷二磷酸醣化因子 (ADP-ribosylation factors, Arfs) 為鳥糞口票呤核苷三磷酸結合蛋白的一種，負責細胞內的囊泡運輸。為了探索酵母菌之第三腺核苷二磷酸醣化因子相似蛋白 (Arf-like 3 protein, Arl3p) 之功能，利用酵母菌雙雜核系統，我們發現一個功能未知的蛋白質可與之結合，命名為第三腺核苷二磷酸醣化相似因子結合蛋白 (Arf-like 3 interacting protein, Ali1p)。Arl3p在細胞中的分布位置與一個位於高基氏體的蛋白質Sft2p幾乎完全相同。Ali1p亦呈現類似高機氏體或之點狀分布。而Arl3p之點狀分布似乎並不需要Ali1p的存在，反之亦然。有趣的是，當Ali1p在細胞中過量表現時，Arl3p的點狀分布位置便不再與Sft2p相同。另外，缺乏這兩個蛋白質的細胞，都顯示對剛果紅(Congo red)的敏感性， 暗示其細胞壁的不完整性。然而，缺乏Arl3p的細胞同時也顯現出一個與細胞壁合成有關的蛋白質Gas1p之不正常的分布，但在缺乏Ali1p的細胞則無此現象。由此推測Arl3p與Ali1p可能經由不同的路徑影響細胞壁之合成。透過合成致死基因的篩選試驗，我們發現許多參與細胞內膜囊泡的運輸的基因與Arl3p之間存在合成致死關係，而這些基因分別負責細胞中不同運輸路徑的調控。利用顏色篩選試驗的方法我們發現Arl3p胺基酸端的前八個胺基酸對於維持其基本功能是必要的。同時破壞ARL3以及COG8這兩個基因將導致一種活體螢光染劑FM4-64在細胞中不正常的堆積，顯示Arl3p與 Cog8p可能參與囊泡的運送或者是液泡的合成。以上研究揭示Arl3p在細胞內膜運輸系統中各種可能扮演的角色。ADP-ribosylation factors (Arfs) are highly conserved small GTP-binding proteins involving vesicular membrane transport. To investigate biological function of a yeast Arf-like protein, Arl3p, yeast two-hybrid screens and synthetic lethal screens of ARL3 were performed. We found a novel protein, named Ali1p (Arf-like 3 interacting protein) that interacts with Arl3p in two-hybrid screen. The in vivo interaction of Ali1p and Arl3p was confirmed by GST-binding assay. Subcellular localization of Arl3p or Ali1p was examined. Arl3p co-localized with late Golgi marker, Sft2p. The Ali1p also showed Golgi-like or endosome-like punctate distribution. Arl3p seems not required for the punctate pattern of Ali1p, and vice versa. Interestingly, Arl3p lost its co-localization with Sft2p when Ali1p was over-expressed. arl3 and ali1 mutants both displayed Congo red sensitivity indicative of cell wall defect. However, arl3, but not ali1 mutant cells exhibited the abnormal distribution of a Gas1p which is involved in cell wall biogenesis, suggesting that Ali1p, different from Arl3p, may participate in distinct pathway with Arl3p to affect the cell wall biogenesis. Synthetic lethal screen of ARL3 was also performed and many candidate genes involved in different vesicular transport pathway were identified. By the colony- sector assay we showed that N-terminal 8 a. a. of Arl3p was essential for its function. Moreover, disruption of both ARL3 and COG8 result in the abnormal accumulation of a vital fluorescence dye, FM4-64, suggesting that Arl3p and Cog8p may be involved in vesicles targeting/fusion or vacuole biogenesis. This study may highlight many possible roles of Arl3p in different trafficking pathways.中文摘要 4 ABSTRACT 5 INTRODUCTION 6 MATERIALS AND METHODS 16 RESULTS 27 I. Arl3p and its interacting protein, Ali1p 27 II. Synthetic lethal screen of ARL3 31 DISCUSSION 37 TABLES AND FIGURES 42 TABLE 1. YEAST STRAINS USED IN THIS STUDY 42 TABLE 2. PRIMERS USED IN THIS STUDY 43 TABLE 3. THE SYNTHETIC LETHAL SCREEN OF ARL3 45 TABLE 4. THE CANDIDATE GENES SYNTHETIC LETHAL WITH ARL3 46 FIGURE 1. STRUCTURE OF SMALL G PROTEINS. 47 FIGURE 2. FUNCTION OF ARF IN MEMBRANE TRAFFIC. 48 FIGURE 3. ARF6-GDP (LEFT) AND ARF6-GTPΓS (RIGHT) ARE SHOWN IN THE SAME ORIENTATION. 49 FIGURE 4. C-TERMINAL REGION OF ALI1P INTERACTED WITH ARL3P IN YEAST TWO-HYBRID SYSTEM. 50 FIGURE 5. GST-ALI1P INTERACTED WITH ARL3P IN VIVO. 51 FIGURE 6. RFP TAGGED ARL3P CO-LOCALIZED WITH LATE GOLGI MARKER SFT2P. 52 FIGURE 7. GFP TAGGED ALI1P DISPLAYED THE PUNCTATE DISTRIBUTION AND THE PATTERN WAS NOT AFFECTED BY DISRUPTION OF ARL3. 53 FIGURE 8. ARL3P DID NOT CO-LOCALIZED WITH ALI1P OVER-EXPRESSED. 54 FIGURE 9. THE PUNCTATE DISTRIBUTION OF ALI1P DID NOT CO-LOCALIZE WITH EITHER GOLGI MARKER EMP47 OR MNN1 55 FIGURE 10. SUBCELLULAR FRACTIONATION OF ALI1P. . 56 FIGURE 11. THE DISTRIBUTION OF ARL3P CHANGED WHEN ALI1P OVER- EXPRESSED. 57 FIGURE 12. DISRUPTION OF ARL3 WOULD AFFECT THE DISTRIBUTION OF GAS1P, BUT DISRUPTION OF ALI1 WOULD NOT 58 FIGURE 13. ARL3 MUTANT CELLS SHOWED CONGO RED SENSITIVITY 59 FIGURE 14. THE TETHERING FACTORS IN VESICLE TRAFFICKING. 60 FIGURE 15. THE CAPABILITY OF DIFFERENT MUTANT FORMS OF ARL3P IN RESCUING THE SECTOR PHENOTYPE. 62 FIGURE 16. DISRUPTION OF BOTH ARL3 AND COG8 WOULD RESULT IN ABNORMAL ACCUMULATION OF FM4-64. 64 REFERENCE 652695191 bytesapplication/pdfen-US第三腺核苷二磷酸醣化因子相似蛋白ARL3 vesicle trafficking Arfotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51374/1/ntu-93-R91448005-1.pdf