本文關(guān)鍵詞：釀酒酵母蛋白質(zhì)激酶Kin2的胞內(nèi)定位、細(xì)胞學(xué)功能及調(diào)控機制的研究　出處：《武漢大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 細(xì)胞極性 囊泡運輸 形態(tài)發(fā)生 Kin1 酵母

【摘要】：釀酒酵母的出芽是一個高度極性化的生長過程,依賴于細(xì)胞骨架的不對稱性組織與定向囊泡運輸。極性生長是大多數(shù)真核生物細(xì)胞分化并實現(xiàn)特定功能的基礎(chǔ),更是真核生物從單細(xì)胞生命體進化為多細(xì)胞生命體的關(guān)鍵,許多蛋白質(zhì)分子被發(fā)現(xiàn)在極性生長中發(fā)揮著調(diào)控作用,其中包括動物細(xì)胞中的MARK/PAR-1激酶,它們在線蟲與果蠅的胚胎發(fā)育過程中體軸的建立、哺乳動物上皮細(xì)胞與神經(jīng)細(xì)胞的細(xì)胞極性建立中有重要作用。釀酒酵母含有兩個與動物細(xì)胞MARK/PAR-1同源的蛋白質(zhì)激酶—Kin1和Kin2,二者在氨基酸序列與細(xì)胞學(xué)功能上極為相似,都參與了囊泡運輸過程的調(diào)控,且在內(nèi)質(zhì)網(wǎng)錯誤折疊蛋白降解途徑中有重要作用,然而,Kin1與Kin2的胞內(nèi)定位還不清楚,之前有報道Kin2位于細(xì)胞質(zhì)中的一些點狀結(jié)構(gòu)中,這與其參與調(diào)控極性生長的功能極不相符。此外,Kin2是否還具有其它細(xì)胞學(xué)功能,其激酶活性是如何被調(diào)控的也不清楚,本論文針對這些問題進行了探索。首先,我們對Kin2的胞內(nèi)定位其介導(dǎo)定位的結(jié)構(gòu)域進行了鑒定。我們在野生型菌株中表達了GFP-Kin2融合蛋白,其表達受KIN2基因自身啟動子的控制且表達元件位于單拷貝質(zhì)粒載體上,我們發(fā)現(xiàn)胞內(nèi)綠色熒光非常弱,幾乎觀察不到。我們于是將表達元件克隆到多拷貝質(zhì)粒載體上,才觀察到了綠色熒光。Kin2定位于極性生長位點,在芽體很小時高度富集于整個芽體的表面；芽體生長到中等大小時,Kin2不再富集于芽體表面；當(dāng)芽體生長至很大,開始進行細(xì)胞質(zhì)分裂時,Kin2富集于芽體與母細(xì)胞的連接處,即芽頸。Kin2定位的這些位點都是細(xì)胞中發(fā)生極性生長的部位,這與其調(diào)控細(xì)胞極性生長的功能非常符合,表明Kin2很可能在這些部位發(fā)揮其調(diào)控極性生長的功能。我們還發(fā)現(xiàn)Kin2具有細(xì)胞膜定位,在整個細(xì)胞膜上都可以觀察到其定位。我們還在另外一種芽殖酵母種類—解脂耶氏酵母中觀察了Kin2同源蛋白—-YlKin1的胞內(nèi)定位,發(fā)現(xiàn)YlKin1具有與Kin2相同的定位。Kin2的極性定位是由分子中哪個結(jié)構(gòu)域所介導(dǎo)的呢?在Kin2分子中,我們鑒定出了兩個介導(dǎo)胞內(nèi)定位的結(jié)構(gòu)域,一個是含有位于其N端的蛋白質(zhì)激酶結(jié)構(gòu)域的定位結(jié)構(gòu)域1(TD1),另一個是含有其C末端KA1 (Kinase-Associated domain 1)結(jié)構(gòu)域的定位結(jié)構(gòu)域2(TD2),它們都能夠介導(dǎo)在極性生長位點的定位,此外,TD2還介導(dǎo)細(xì)胞膜定位。我們的研究發(fā)現(xiàn)TD1對于Kin2參與調(diào)控囊泡運輸過程具有重要作用,不能正常定位的Kin2之N端片段不能挽救sec1-1、sec2-41和sec15-1突變體的生長缺陷,表明Kin2在胞內(nèi)極性生長位點的定位對于其調(diào)控囊泡運輸是必需的。由于TD2對于囊泡運輸具有負(fù)調(diào)控作用,其缺失會增強Kin2的功能,推測其介導(dǎo)的在整個細(xì)胞表面的定位可能是用于調(diào)控其它細(xì)胞學(xué)過程,如細(xì)胞壁的合成(見下段描述)。其次,我們對Kin2可能具有的其它細(xì)胞學(xué)功能進行了研究。我們構(gòu)建了kin1△ kin2△雙缺失菌株,但是,該菌株并不具有任何生長或細(xì)胞形態(tài)缺陷。我們于是在細(xì)胞中對Kin2進行了過量表達,Kin2的過量表達并不抑制細(xì)胞生長,但是,細(xì)胞形態(tài)出現(xiàn)一定程度的缺陷—芽體變長與形成細(xì)胞簇,尤其是在septin細(xì)胞骨架組織存在缺陷的gin4A菌株中該形態(tài)缺陷尤為顯著,胞內(nèi)septin細(xì)胞骨架的組織以及細(xì)胞壁幾丁質(zhì)的沉積出現(xiàn)紊亂,細(xì)胞對破壞細(xì)胞壁合成的calcofluor及破壞細(xì)胞膜結(jié)構(gòu)的去垢劑SDS都較未過量表達Kin2的對照菌株敏感,表明Kin2可能參與對septin細(xì)胞骨架的組織與細(xì)胞壁的調(diào)控。TD1和TD2兩個定位結(jié)構(gòu)域都對這個過程都有所貢獻。過量表達Kin2的N端TD1結(jié)構(gòu)域還能夠抑制細(xì)胞生長,細(xì)胞變大變圓,部分喪失極性。通過酵母雙雜交,我們發(fā)現(xiàn)Kin2能夠與Tos1、septin亞基Cdc11以及極性小體(polarisome)成員Pea2相互作用,Kin2的C端片段還能夠與Pea2發(fā)生雙分子熒光互補(BiFC)相互作用。Tosl是一個與細(xì)胞壁共價結(jié)合的蛋白,我們推測Kin2可能通過Tosl調(diào)控細(xì)胞壁合成。由于極性小體參與septin的組織,我們推測Kin2可能通過Pea2與septin亞基Cdc11相互作用而調(diào)控septin組織。再次,通過酵母雙雜交文庫篩選,我們發(fā)現(xiàn)Kin2的C端區(qū)段能夠與Rho GTP酶Rho3相互作用,且Kin2偏愛與結(jié)合GTP的激活型Rho3相互作用。通過GST pull-down與雙分子熒光互補,我們也檢測到Kin2能夠與Rho3在體內(nèi)相互作用,且相互作用發(fā)生在極性生長位點,推測Rho3可能是一個作用于Kin2上游的調(diào)控分子,幫助解除Kin2分子C端對激酶結(jié)構(gòu)域活性的抑制,對于激活Kin2的激酶活性有作用。我們發(fā)現(xiàn)提高Pho3Q74L的表達量能夠促進過量表達Kin2造成的對septin組織的破壞,暗示著Rho3可能具有促進Kin2活性的功能,然而,我們在sec3-2、sec1-1和sec2-41等分泌功能缺陷的突變體中多拷貝表達Rho3后并沒有發(fā)現(xiàn)Rho3對Kin2調(diào)控囊泡運輸過程的功能具有促進作用,這一可能性是否存在尚需要其它實驗來檢驗。我們還篩選到14-3-3蛋白Bmhl能夠與Kin2相互作用,且Bmh1作用于Kin2的N端,我們推測Bmh1可能對于Kin2活性狀態(tài)的維持有著重要的作用。Kin2同源蛋白在真菌中廣泛存在,在一些致病性真菌,如新型隱球酵母、稻瘟病菌與禾谷鐮刀菌中,Kin2同源蛋白是重要的致病因子之一,本課題研究成果對于揭示這些真菌的致病性機制具有重要的科學(xué)意義。
[Abstract]:The budding yeast is the growth process of a highly polarized, depend on cytoskeletal organization and directional asymmetry in vesicular transport. Polar growth is the basis of most eukaryotic cell differentiation and the realization of specific functions, is the key to eukaryotes from single celled organism for the evolution of multicellular organisms. Many proteins are found in polar growth plays a regulatory role in animal cells, including MARK/PAR-1 kinase, a body axis in the process of their nematode and Drosophila embryo development, mammalian epithelial cells and nerve cells have an important role in the establishment of cell polarity in Saccharomyces cerevisiae. And animal cells containing two MARK/PAR-1 homologous protein kinase Kin1 and Kin2, two in the amino acid sequence and the cytological function is very similar, are involved in the regulation of vesicle transport process, and the error in the endoplasmic reticulum There is an important role in the folding pathway for protein degradation. However, Kin1 and Kin2 of the intracellular localization is not clear before, Kin2 has been reported in some punctate structures in the cytoplasm, it is involved in the regulation of polar growth function is not very consistent. In addition, Kin2 also has other cell function, its kinase activity is how regulation is not clear, this paper explores the problems. Firstly, we Kin2 on the intracellular localization of domain mediated localization were identified. The expression of GFP-Kin2 fusion protein in the wild-type strain, its expression is controlled by KIN2 gene promoter and its expression element in a single copy the plasmid vector, we found that intracellular green fluorescence is very weak, almost not observed. We then cloned into the expression element of multicopy plasmid vector, green fluorescence was observed in.Kin2 polar growth potential In the bud, is highly enriched in the surface h bud bud; growth to moderate size, Kin2 no longer enriched in the bud bud growth to the surface; when large, began cytokinesis, at the junction of Kin2 enriched in the bud and mother cell, the location of the bud neck.Kin2 of these sites are polar growth occurs in the cell, and the regulation of cell polarity growth function is consistent with, that Kin2 may exert its regulation in these parts of the polar growth function. We also found that Kin2 has cell membrane localization, we can observe the position in the whole cell membrane. We are also a budding yeast species - Yarrowia lipolytica was observed in Kin2 homologous protein -YlKin1 localization within the cell, found that the polar localization of YlKin1 with.Kin2 and Kin2 location of the same domain consists of molecules which are mediated in Kin? 2 molecules, we have two domain mediated intracellular localization and identification, one is located in the localization domain containing protein kinase domain of the N at the end of 1 (TD1), the other is a KA1 containing its C terminal (Kinase-Associated domain 1) localization domain domain 2 (TD2), they are able to mediate in the polar growth site localization, in addition, TD2 also mediates cell membrane localization. We found that TD1 plays an important role in Kin2 is involved in the regulation of vesicle transport process, not normal positioning of the Kin2 N fragment can save sec1-1, growth defects in sec2-41 and sec15-1 mutants, that Kin2 in cell polarity growth site localization is required for the regulation of vesicular transport. Due to TD2 for vesicle transport has a negative regulatory role, and its absence will enhance the function of Kin2, presumably mediated in the positioning of the entire cell surface may be used to control the The cellular processes, such as cell wall synthesis (see description). Secondly, we may have other cellular functions of Kin2 were studied. We construct a kin1 Delta kin2 delta double mutant strain, but the strain does not have any growth or morphological defects. So we were carried out on the Kin2 overexpression of Kin2 overexpression did not inhibit cell growth, but cell morphological defects - degree of variable length and bud formation of cell clusters, especially strain gin4A defects in the septin cytoskeleton organization in the form of defects is particularly significant, intracellular septin cytoskeletal organization and cell wall deposition of chitin disorder the destruction of Calcofluor cells, detergent and destroy the structure of cell membrane and the cell wall synthesis agent SDS is not sensitive to overexpression of the control strain Kin2, indicating that Kin2 may be involved in the septin Tissue and cell wall cytoskeleton in the regulation of.TD1 and TD2 two positioning domain of this process to contribute. Overexpression of N terminal TD1 domain of Kin2 can inhibit cell growth, cells became round, partial loss of polarity. By yeast two hybrid system, we found that Kin2 and Tos1 can. The septin subunit of Cdc11 and polar bodies (polarisome) members of the Pea2 interaction, Kin2 C fragment can also undergo bimolecular fluorescence complementation and interaction of Pea2 (BiFC).Tosl is a cell wall protein covalent binding, we speculate that Kin2 may be regulated by Tosl cell wall synthesis. Because the polar bodies involved in the organization septin, we speculate that Kin2 may through the Pea2 and septin subunits of Cdc11 interaction and regulation of septin organization. Once again, the yeast two hybrid library screening, we found that Kin2 can C Rho GTP Rho3 end section and enzyme interaction, and Kin2 With the preference and the activation of GTP Rho3 interaction. Through the GST pull-down and bimolecular fluorescence complementation, we detected that Kin2 and Rho3 can interact in vivo, and the interaction occurs at the polar growth sites, suggesting that Rho3 may be a regulatory role in the upstream of Kin2 molecules, Kin2 molecules help relieve C end inhibition of the kinase domain activity, plays a role in the activation of Kin2 kinase activity. We found that increasing the expression of Pho3Q74L can promote the expression of Kin2 of septin caused by excessive tissue damage, suggesting that Rho3 could promote the activity of Kin2, however, we are in sec3-2, sec1-1 and sec2-41 multi copy secretion defect the expression of Rho3 mutants and found no Rho3 on Kin2 regulation of vesicle transport process has the function of promoting effect, whether this possibility needs other experiments to test. We also screen Elected to the 14-3-3 Bmhl protein can interact with Kin2, and the effect of Bmh1 on the Kin2 N terminal, we speculate that Bmh1 may maintain for Kin2 activity plays an important role in.Kin2 homologous protein existed in fungi, in some pathogenic fungi, such as Cryptococcus, Magnaporthe grisea and Fusarium graminearum. Kin2, a homologous protein is one of the important virulence factors, this research has important scientific significance to reveal the mechanism of pathogenic fungi.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q936

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