李芳仁臺灣大學：分子醫學研究所Liu, Ya-WenYa-WenLiu劉雅雯2007-11-262018-07-092007-11-262018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/51359腺嘌呤核苷二磷酸核醣化因子(ADP-ribosylation factor, ARF)為Ras致癌基因蛋白家族的一支，屬於鳥糞嘌呤核苷三磷酸結合蛋白的一種。在各種不同的真核生物都有ARF家族成員的分布，負責著細胞內的囊泡運輸、磷脂酶的活化、參與磷脂三激酶的訊息調控等等的功能。ARF家族成員可分為ARF與ARF-相似蛋白(ARL)兩大類。目前雖然已知有許多ARL的存在但對其功能卻仍幾乎是一無所知。本文利用酵母菌為模式研究ARL1蛋白在細胞中所扮演的角色。利用螢光染色法，我們發現ARL1蛋白分布在高基氏體上，並利用酵母菌雙雜合篩選法找出與Arl1p蛋白直接作用的蛋白質Imh1p以及Gcs1p。從接下來的功能性探討，我們證明了Imh1p是Arl1p的下游作用基因受其調控，而Gcs1p 是Arl1p的上游調控者會活化其鳥糞嘌呤核苷三磷酸水解能力並繼而影響Arl1p在細胞中的分布。另外，在囊泡運輸路徑的研究中，我們發現ARL1蛋白會參與在醣基化磷脂醯肌醇(glycosylphosphatidylinositol) 連結蛋白Gas1p從高基氏體到細胞膜的運送。無論是ARL1蛋白的上游調控基因或下游作用基因都會影響Gas1p。因此，我們的研究發現了部份ARL1蛋白的結合蛋白及其結合功能以及Arl1p在酵母菌中所參與的囊泡運輸路徑。進一步分析與探討其他與其作用的蛋白質將會使我們對囊泡運輸的分子機轉有更深一層的了解。ADP-ribosylation factors (ARFs) are highly conserved small GTP-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and have an important role in vesicular transport. Several ARF-like proteins (ARLs) have been cloned from different organisms but the biological functions of ARLs remain largely unknown. In this dissertation, an ARL protein, Arl1p, of yeast Saccharomyces cerevisiae was characterized. Yeast ARL1 encodes a protein that is structurally related to human, rat, and Drosophila ARL1 proteins. Here we identified two interacting proteins of Arl1p, Imh1p and Gcs1p, the former is a tethering factor and the latter is an ARFGAP. From several in vitro and in vivo analysis, we demonstrated that Imh1p is a downstream effector of Arl1p and is recruited by the proper protein-protein interaction; on the other hand, Gcs1p exhibits GAP activity toward Arl1p and is able to regulate subcellular localization of Arl1p thus indicates that Gcs1p is a GAP for Arl1p. To the functional analysis, we found that Arl1p is involved in the anterograde transport from the Golgi to cell surface of the glycosylphosphatidylinositol (GPI)-anchored plasma membrane-resident protein Gas1p, but not those cell wall-localized GPI-anchored proteins Crh1p, Crh2p, and Cwp1p, or non-GPI-anchored plasma membrane-protein Gap1p. We also show that regulators of Arl1p: Sys1p, Arl3p, and Gcs1p; and effector Imh1p, all participate in the transport of Gas1p. From these results, not only the first ARL GAP is identified, but the first endogenous cargo molecule of yeast Arl1p is defined.Abstract---------------------------------------------------4 中文摘要---------------------------------------------------5 Abbreviations----------------------------------------------6 Introduction-----------------------------------------------7 Materials and Methods-------------------------------------20 Results I. Localization of Arl1p--------------------------------39 II. Interacting proteins of Arl1p-----------------------40 III. Functions of Arl1p in vesicular transport----------46 Discussion------------------------------------------------56 Tables Table 1. Yeast strains used in this study--------------66 Table 2. Primers used in this study--------------------67 Figures Figure 1. Regulation of ARF activity by GEPs and GAPs ----70 Figure 2. Structure of ARF1 ------------------------------71 Figure 3. ARFGAP sequence and structure of the ARF1t-ARFGAP complex-----------------------------------------72 Figure 4. Model for general mechanism of transport vesicle budding-----------------------------------------73 Figure 5. Pathway of Arl1p recruitment to Golgi membranes-74 Figure 6. Arl1p partially colocalizes with GFP-Sft2p------75 Figure 7. Localization of Arl1p on trans-Golgi is nucleotide dependent---------------------------------------76 Figure 8. Interaction of ARLs or ARFs with Imh1p----------77 Figure 9. In vitro and in vivo interactions of Arl1p and Imh1p-------------------------------------------78 Figure 10. Arl1p is required for the Golgi association of Imh1p------------------------------------------79 Figure 11.Interaction of Arl1Q72Ld17N with Gcs1p in two-hybrid assay--------------------------------80 Figure 12. Arl1p interacts with Gcs1p in vitro and in vivo81 Figure 13. GAP activity of Gcs1p for Arl1p in vitro-------82 Figure 14. More Arl1p is present in late Golgi of gcs1 mutant than in wild type cells------------------------83 Figure 15. More Arl1-mRFP is present in late Golgi of gcs1 mutant than wild type--------------------------84 Figure 16. Overexpression of Gcs1-myc caused dissociation of endogenous Arl1p from trans-Golgi--------------85 Figure 17. Overexpression of Gcs1-GFP, but not Gcs1-zn-GFP, caused dissociation of endogenous Arl1p from trans-Golgi------------------------------------86 Figure 18. GFP-Imh1p substantially loses its Golgi association in arl1- and gcs1-null cells-------87 Figure 19. Constitutive active and inactive mutant forms of Arl1p did not complement the loss of Arl1p-----88 Figure 20. Arl1p is not directly involved in the recycling of GFP-Snc1p and the processing of vacuolar proteins -----------------------------------------------89 Figure 21. Arl1p is required for the surface targeting of GFP-GPI (Gas1p)--------------------------------90 Figure 22. Arl1p is involved in transport of Gas1p from the late Golgi to plasma membrane -----------------91 Figure 23. Sys1p is involved in transport of Gas1p -------93 Figure 24. Over-expression of active Arl1p rescues Congo red hyperseneitivity of arl3 mutant ---------------94 Figure 25. Subcellular distribution of endogenous Gas1p --95 Figure 26. Deletion of IMH1, but not YPT6, affects transport of Gas1p to the plasma membrane----------------96 Figure 27. Arl1p regulates the integrity of the cell wall and localization of Gas1p in a GTP-dependent manner97 Figure 28. Arl1Q72L localizes to the late Golgi in arl3 mutant-----------------------------------------99 Figure 29. The model for Arl1p activity-----------------100 Figure 30. The model for aberrant Arl1p activity --------101 Reference-------------------------------------------------1026434540 bytesapplication/pdfen-US腺嘌呤核苷二磷酸核醣化因子 囊泡運輸ARF vesicular trafficking[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51359/1/ntu-95-F89448006-1.pdf