李芳仁臺灣大學：分子醫學研究所李思緯Lee, Szu-WeiSzu-WeiLee2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51334腺嘌呤核苷二磷酸核醣化因子(ADP-ribosylation factors, ARFs)屬於演化上具高度保留性的小鳥糞嘌呤核苷三磷酸結合蛋白(small GTP-binding proteins)，在真核細胞內胞器之間的囊泡生成與傳遞過程中扮演重要的角色。雖然酵母菌的第三腺嘌呤核苷二磷酸核醣化因子(Arf3p)與哺乳動物的第六腺嘌呤核苷二磷酸核醣化因子(ARF6)最為相似，但卻沒有參與流相、膜內化或交配型受體媒介之胞飲作用，反而在酵母菌出芽生殖時不對稱結構(極性；polarity)的建立過程中發揮功能。為了更深入探究Arf3p的生物功能，我們採用功能性研究及遺傳學探討的方式。先前大規模的酵母菌雙雜合(yeast two-hybrid)分析結果指出第二出芽位置選擇蛋白(bud site selection; Bud2p)與Arf3p之間有相互作用，而本研究結果顯示於活的有機體內(in vivo) Bud2p以仰賴鳥糞嘌呤核苷三磷酸(GTP)的形式與Arf3p有專一性的結合。Bud2p在細胞中的分布不受酵母菌第三腺嘌呤核苷二磷酸核醣化因子基因(ARF3)剔除所影響，反之亦然。然而，非極化且大量表現之Arf3p及其永久活化型(Arf3Q71L)會因極化且大量表現之Bud2p而集中在母體與芽體連接處，這顯示了在活的細胞內Bud2p確實與Arf3p有作用。雖然Bud2p為第一出芽位置選擇蛋白(Bud1p)的GTP水解活化蛋白(GTPase-activating protein; GAP)，但其可能是Arf3p的調節者而不是GAP。此外，在高溫的情況下，分別剔除這兩個基因皆會影響早期肌動蛋白在芽體的聚集，但同時剔除這兩個基因並不會加劇此顯型，意味著Bud2p和Arf3p參與於相同的途徑來間接調控肌動蛋白細胞骨架的結構。再者，兩者皆非環化酶結合蛋白無效等位基因第一抑制者[suppressor of the null allele of CAP (cyclase-associated protein); Snc1p]及第三幾丁質合成酶(chitin synthase 3; Chs3p)的運輸所必需。Bud2p與Arf3p之間真正的關係尚待釐清，而Arf3p與胞飲作用之機轉的相互關係亦需進一步的探討。ADP-ribosylation factors (ARFs) are highly conserved small GTP-binding proteins and are critical components of vesicular trafficking in eukaryotic cells. Yeast Arf3p, in spite of its similarity to mammalian homologue ARF6, is not required for fluid-phase, membrane internalization or mating-type receptor-mediated endocytosis; instead, it is involved in polarity development. To further explore the biological functions of Arf3p, functional and genetic studies were adopted. Bud2p, which was reported to show an interaction with Arf3p in large-scale yeast two-hybrid analysis, specifically interacts with Arf3p in a GTP-dependent manner in vivo. The subcellular localization of Bud2p is not affected by ARF3 disruption, and vice versa. However, nonpolarized overexpressed Arf3p and Arf3Q71L are concentrated at the mother-bud junction by polarized overexpressed Bud2p, indicating that Bud2p also interacts with Arf3p in living cells. Although BUD2 encodes a GAP for Bud1p, it may not be a GAP for Arf3p. BUD2 disruption as well as ARF3 disruption exhibits early actin patch depolarization at higher temperatures, and combination of arf3 and bud2 deletions does not exacerbate the phenotype, implying that Bud2p and Arf3p contribute a role in the same pathway indirectly regulating actin cytoskeleton organization. Moreover, neither Arf3p nor Bud2p is required for Snc1p and Chs3p trafficking. The precise relationship between Bud2p and Arf3p remains to be elucidated. Besides, an interaction between Arf3p and the endocytic machinery, in which Lsb5p and Ysc84p were shown to be together involved, needs to be determined.中文摘要 4 Abstract 5 Introduction 6 Small GTP-binding proteins 6 Structure 7 A role as molecular switches 8 ADP-ribosylation factors (ARFs) 9 Structure of ARFs 9 Guanine nucleotide exchange factors 11 GTPase-activating proteins 11 Effectors of ARFs 12 Mammalian ARFs 12 ARF function in vesicle formation 14 ARF function in actin remodeling 15 Yeast ARFs 16 Materials and Methods 18 Results 29 I. Arf3p and its interacting protein, Bud2p 29 Bud2p interacts with Arf3p in yeast two-hybrid analysis 29 Bud2p interacts with Arf3p in vivo 29 Arf3p is not required for Bud2p localization, and vice versa 30 Bud2p may not be a GTPase-activating protein for Arf3p 32 Arf3-mRFP and Arf3Q71L-mRFP are enriched to the mother-bud junction by polarized GFP-Bud2 and colocalized with GFP-Bud2 at the neck and the plasma membrane 33 BUD2 is not synthetic lethal with ARF3 34 Both Bud2p and Arf3p are indirectly involved in actin patch polarization 34 Neither Arf3p nor Bud2p participates in trafficking of Snc1p and Chs3p 35 II. Genetic studies of ARF3 38 Synthetic lethal screen of ARF3 38 Neither LSB5 nor YSC84 has genetic interactions with ARF3 40 Discussion 42 References 57 Table 1. Yeast strains used in this study 69 Table 2. Primers used in this study 71 Table 3. Antibodies used in this study 73 Table 4. Synthetic lethal screenings performed in this study …………74 Figure Legends …………………………………………………75 Figures ……………………………………………………………81 Figure 1: Structure of small GTP-binding proteins …………………………81 Figure 2: The interswitch toggle of human ARF6 ………………………………82 Figure 3: Function of ARF in membrane traffic ………………………………83 Figure 4: Schematic of feedforward and feedback loops involving ARF and phosphoinositides …………………………………………………………84 Figure 5: Interactions between Bud2p and Arf3p in yeast two-hybrid analysis ……………………………………………………………………85 Figure 6: In vivo interactions between Bud2p and Arf3p ……………………86 Figure 7: ARF3 disruption does not affect the localization of overexpressed GFP-Bud2 …………………………………………………………………87 Figure 8: Subcellular localization of Arf3-GFP and it mutants is not affected by BUD2 disruption ……………………………………………………..88 Figure 9: Bud2p overexpression does not affect the subcellular localization of Arf3-GFP ………………………………………………………………89 Figure 10: Arf3-mRFP and Arf3Q71L-mRFP are enriched at the mother-bud junction by polarized GFP-Bud2 and colocalized with GFP-Bud2 at the neck and the plasma membrane ……………………90 Figure 11: ARF3 or BUD2 disruption does not affect the growth rate …….92 Figure 12: Both Bud2p and Arf3p are indirectly involved in actin patch polarization ……………………………………………………………….93 Figure 13: ARF3 disruption does not affect FM4-64 uptake at 34oC ……………94 Figure 14: Subcellular distribution of Snc1-GFP is not affected by ARF3 or BUD2 disruption ………………………………………………………………95 Figure 15: Chs3-GFP distribution is not affected by ARF3 or BUD2 disruption ………………………………………………………………96 Figure 16: Neither LSB5 nor YSC84 has a genetic interaction with ARF3 ………97 Figure 17: BY4741 lsb5 ysc84 double deletion cells exhibit no defects in FM4-64 uptake and transport, but some fragmentation of vacuoles ………98 Appendix A ……………………………………………………………99 Appendix B …………………………………………………………1003026572 bytesapplication/pdfen-US腺嘌呤核苷二磷酸核醣化因子ADP-ribosylation factorotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51334/1/ntu-94-R92448002-1.pdf