李芳仁Lee, Fang-Jen臺灣大學:分子醫學研究所何瑞芝Ho, Jui-ChihJui-ChihHo2010-05-042018-07-092010-05-042018-07-092008U0001-2507200813111500http://ntur.lib.ntu.edu.tw//handle/246246/178668腺嘌呤核苷二磷酸核醣化因子 (ADP-ribosylation factors, ARFs) 在真核細胞內胞器之間的囊泡生成與傳遞扮演重要的角色。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，但卻不參與胞飲作用，反而在酵母菌出芽生殖時極性建立與肌動蛋白的極化現象發揮調控功能。欲進一步探討 Arf3p 之功能，本研究中發現，酵母菌在含半乳糖的培養基中發展絲狀生長與在固態平板培養基中發展侵入生長皆需要 Arf3p 的存在。適量表現帶有綠色螢光標記的 Arf3p 融合蛋白或無標記可嵌入染色體的 Arf3p，皆僅能部份救回 arf3 突變株的絲狀生長缺陷。然而，表現任一種 Arf3p 皆無法救回arf3 突變株的侵入生長缺陷。在雙套異型合子的 arf3 突變株中，絲狀生長的缺陷則可被成功救回。近期研究發現酵母菌的第六腺嘌呤核苷二磷酸核醣化因子之交換因子第一相似蛋白 (Yel1p, Yeast EFA6-like-1) 功能為 Arf3p 的一個鳥糞嘌呤核苷酸交換因子。利用酵母菌雙雜交系統證實 Arf3p 與 Yel1p 之交互作用。近期研究也對酵母菌第三腺嘌呤核苷二磷酸核醣化因子結合蛋白 (Afi1p, Arf3p-interacting protein 1) 之功能有深入探討。欲研究此二 Arf3p 結合蛋白，Yel1p 與 Afi1p，對酵母菌絲狀生長之影響，由本研究結果顯示 Afi1p 與 Arf3p 皆對絲狀生長以及侵入生長發揮功能，Yel1p僅對侵入生長有明顯影響。利用嵌入型的 Afi1p 僅能部份救回 afi1 突變株的絲狀生長缺陷，胺基端帶有點突變的 Afi1p (Afi138KLGP4A) 喪失與 Arf3p 結合之能力，則完全無法救回 afi1 突變株的絲狀生長缺陷。在能表現絲狀生長之菌株 Σ1278b 單一突變 arf3、afi1 或 yel1 都會影響肌動蛋白極化現象。欲進一步探討 Afi1p 之功能，截短羧酸端 20 個胺基酸之 Afi1p 不影響其本身或 Arf3p 在細胞膜上之極化分布，但卻會影響肌動蛋白之極化分布。本研究結果顯示，Afi1p 與 Arf3p 皆對絲狀生長以及侵入生長發揮功能，Yel1p 則只影響侵入生長。此外，Arf3p 與其結合蛋白，Yel1p 與 Afi1p，都在維持肌動蛋白之極化方面扮演重要角色。由本研究結果更進一步了解 Afi1p 之功能在利用胺基端與 Arf3p 有交互作用，並且共同作用於同一調控途徑，而羧酸端 Afi1p 可能不參與調控 Arf3p 之極化分布。欲了解 Arf3p 與 Afi1p 如何參與調控酵母菌絲狀生長與侵入生長則須待進一步探討。ADP-ribosylation factors (ARFs) are ubiquitous regulators of vesicular membrane traffic. Yeast Arf3p, which is most similar to mammalian ARF6 is not required for endocytosis but involves in polarity development and actin organization. In this study, the role of Arf3p has been further characterized, and it has been shown that Arf3p is required for not merely filamentous growth in galactose-containing medium but also invasive growth on rich medium plate. Moderate expression of either Arf3-GFP or non-tagged integrated Arf3p could partially rescue the filamentous defect observed in arf3 mutant. However, the invasive defect of arf3 could not be rescued by any ARF3 constructs. In addition, the invasive defect could be fully restored in diploid heterozygous arf3 mutant containing wild-type allele of ARF3. Recent study for Arf3p has shown Yel1p (Yeast EFA6-like-1) acts as one of the guanine nucleotide exchange factors (GEFs) for Arf3p. The physical interaction between Arf3p and Yel1p was further confirmed by yeast two-hybrid analysis in this study. Moreover, another Arf3p-interacting protein, Afi1p (Arf3p-interacting protein 1) has been identified in our previous study. Further examination of the two Arf3p-interacting proteins, Yel1p and Afi1p, revealed that Afi1p was required for both filamentous and invasive growth; but Yel1p only involves in invasiveness of yeast cells. Integrated form of Arf3p-interacting Afi1p could partially rescue the filamentous defect in afi1. N-terminal mutant of Afi1p (Afi138KLGP4A) which did not interact with Arf3p could not rescue the filamentous defect in afi1. In this filamentous strain of Σ1278b background, arf3, afi1, or yel1 deletion mutants exhibited defect on actin patch polarization, consistent with previous studies. To further determine the function of Afi1p, C-terminal truncation of Afi1p revealed that lacking of C-terminal 20 amino acids did not affect either Afi1p itself polarization or Arf3p polarization but actin patches could not polarize properly. In short, this study demonstrated that Arf3p and Afi1p are both indispensable for filamentous and invasive growth, but Yel1p is not. Arf3p and its interacting proteins, Afi1p and Yel1p, all involve in regulation for actin cytoskeleton organization. From this study, N-terminal Afi1p which is critical for interacting with Arf3p may act in the same pathway with Arf3p, while C-terminal Afi1p may have other functions than maintaining Arf3p polarization. The detailed mechanism of Arf3p and Afi1p to regulate filamentous and invasive growth requires further investigation.中文摘要 IIIbstract IVbbreviations VIntroduction 1aterials and Methods 13esults 20rf3p is required for filamentous and invasive growth 20rf3-GFP can partially rescue defect of cell elongation, not invasion, in arf3 mutant 21ntegrated form of Arf3p can partially rescue defect of cell elongation, not invasion, in arf3 mutant 21nvasive defect of arf3 can be complemented by wild-type allele in heterozygous arf3 mutants 21rf3p with endogenous expression level may result in partial rescue of filamentous defect 22rf3p and Yel1p have physical interaction 23fi1p affects both filamentous and invasive growth, but Yel1p is only required for invasive growth 23-terminal conserved region of Afi1p is important for filamentous growth 25ctin patches polarization is disrupted in arf3, afi1, or yel1 mutants 26fi1p with C-terminally truncated 20 amino acids is able to polarize on plasma membrane 27-terminally truncated Afi1p does not abolish polarization of Arf3p on plasma membrane but affects actin patch polarization 28iscussion 30ables 36able 1. Yeast strains used in this study 36able 2. Primers used in this study 38able 3. Plasmids used in this study 40able 4. Antibodies used in this study 41igures 42igure 1 Structure of small GTP-binding proteins.. 42igure 2 Regulation of ARF activity by GEFs and GAPs. 44igure 3 Schematic of feedforward and feedback loops involving Arf and phosphoinositides. 45igure 4 Arf3p is required for filamentous and invasive growth. 46igure 5 Arf3-GFP can partially rescue defect of cell elongation in arf3 mutant but cannot rescue its invasive defect. 47igure 6 Integrated non-tagged form of Arf3p can partially rescue defect of cell elongation. 48igure 7 Both filamentous and invasive defects can be fully restored in diploid heterozygous arf3 mutants. 49igure 8 Moderate expression of exogenous Arf3p may result in partial rescue of filamentous defect. 50igure 9 Arf3p and Yel1p have physical interaction.. 52igure 10 Effect on actin patch polarization in yel1 mutant. 54igure 11 The Arf3p-interacting protein Afi1p affects both filamentous and invasive growth, but Yel1p affects only filamentous growth………………..54igure 12 Effect of KLGP4A mutant of Afi1p on actin patch polarization.. 56igure 13 N-terminal conserved region of Afi1p is important for filamentous growth.………………………………………………………………...………..56igure 14 Actin patches polarization is disrupted in arf3, afi1, or yel1 mutants.. 57igure 15 Afi1p with C-terminally truncated 20 amino acids does not affect its polarization on plasma membrane. 58igure 16 Afi1p with C-terminal truncation does not abolish Arf3p polarization.. 59igure 17 C-terminus of Afi1p involves in actin patch polarization. 60eferences 61application/pdf3127988 bytesapplication/pdfen-US腺嘌呤核苷二磷酸核醣化因子肌動蛋白極化現象絲狀生長ADP-ribosylation factoractin polarizationfilamentous growthhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178668/1/ntu-97-R95448009-1.pdf