酿酒酵母蛋白质激酶Kin2的胞内定位、细胞学功能及调控机制的研究

发布时间：2018-01-08 02:21

  本文关键词：酿酒酵母蛋白质激酶Kin2的胞内定位、细胞学功能及调控机制的研究　出处：《武汉大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 细胞极性 囊泡运输 形态发生 Kin1 酵母

【摘要】：酿酒酵母的出芽是一个高度极性化的生长过程,依赖于细胞骨架的不对称性组织与定向囊泡运输。极性生长是大多数真核生物细胞分化并实现特定功能的基础,更是真核生物从单细胞生命体进化为多细胞生命体的关键,许多蛋白质分子被发现在极性生长中发挥着调控作用,其中包括动物细胞中的MARK/PAR-1激酶,它们在线虫与果蝇的胚胎发育过程中体轴的建立、哺乳动物上皮细胞与神经细胞的细胞极性建立中有重要作用。酿酒酵母含有两个与动物细胞MARK/PAR-1同源的蛋白质激酶—Kin1和Kin2,二者在氨基酸序列与细胞学功能上极为相似,都参与了囊泡运输过程的调控,且在内质网错误折叠蛋白降解途径中有重要作用,然而,Kin1与Kin2的胞内定位还不清楚,之前有报道Kin2位于细胞质中的一些点状结构中,这与其参与调控极性生长的功能极不相符。此外,Kin2是否还具有其它细胞学功能,其激酶活性是如何被调控的也不清楚,本论文针对这些问题进行了探索。首先,我们对Kin2的胞内定位其介导定位的结构域进行了鉴定。我们在野生型菌株中表达了GFP-Kin2融合蛋白,其表达受KIN2基因自身启动子的控制且表达元件位于单拷贝质粒载体上,我们发现胞内绿色荧光非常弱,几乎观察不到。我们于是将表达元件克隆到多拷贝质粒载体上,才观察到了绿色荧光。Kin2定位于极性生长位点,在芽体很小时高度富集于整个芽体的表面；芽体生长到中等大小时,Kin2不再富集于芽体表面；当芽体生长至很大,开始进行细胞质分裂时,Kin2富集于芽体与母细胞的连接处,即芽颈。Kin2定位的这些位点都是细胞中发生极性生长的部位,这与其调控细胞极性生长的功能非常符合,表明Kin2很可能在这些部位发挥其调控极性生长的功能。我们还发现Kin2具有细胞膜定位,在整个细胞膜上都可以观察到其定位。我们还在另外一种芽殖酵母种类—解脂耶氏酵母中观察了Kin2同源蛋白—-YlKin1的胞内定位,发现YlKin1具有与Kin2相同的定位。Kin2的极性定位是由分子中哪个结构域所介导的呢?在Kin2分子中,我们鉴定出了两个介导胞内定位的结构域,一个是含有位于其N端的蛋白质激酶结构域的定位结构域1(TD1),另一个是含有其C末端KA1 (Kinase-Associated domain 1)结构域的定位结构域2(TD2),它们都能够介导在极性生长位点的定位,此外,TD2还介导细胞膜定位。我们的研究发现TD1对于Kin2参与调控囊泡运输过程具有重要作用,不能正常定位的Kin2之N端片段不能挽救sec1-1、sec2-41和sec15-1突变体的生长缺陷,表明Kin2在胞内极性生长位点的定位对于其调控囊泡运输是必需的。由于TD2对于囊泡运输具有负调控作用,其缺失会增强Kin2的功能,推测其介导的在整个细胞表面的定位可能是用于调控其它细胞学过程,如细胞壁的合成(见下段描述)。其次,我们对Kin2可能具有的其它细胞学功能进行了研究。我们构建了kin1△ kin2△双缺失菌株,但是,该菌株并不具有任何生长或细胞形态缺陷。我们于是在细胞中对Kin2进行了过量表达,Kin2的过量表达并不抑制细胞生长,但是,细胞形态出现一定程度的缺陷—芽体变长与形成细胞簇,尤其是在septin细胞骨架组织存在缺陷的gin4A菌株中该形态缺陷尤为显著,胞内septin细胞骨架的组织以及细胞壁几丁质的沉积出现紊乱,细胞对破坏细胞壁合成的calcofluor及破坏细胞膜结构的去垢剂SDS都较未过量表达Kin2的对照菌株敏感,表明Kin2可能参与对septin细胞骨架的组织与细胞壁的调控。TD1和TD2两个定位结构域都对这个过程都有所贡献。过量表达Kin2的N端TD1结构域还能够抑制细胞生长,细胞变大变圆,部分丧失极性。通过酵母双杂交,我们发现Kin2能够与Tos1、septin亚基Cdc11以及极性小体(polarisome)成员Pea2相互作用,Kin2的C端片段还能够与Pea2发生双分子荧光互补(BiFC)相互作用。Tosl是一个与细胞壁共价结合的蛋白,我们推测Kin2可能通过Tosl调控细胞壁合成。由于极性小体参与septin的组织,我们推测Kin2可能通过Pea2与septin亚基Cdc11相互作用而调控septin组织。再次,通过酵母双杂交文库筛选,我们发现Kin2的C端区段能够与Rho GTP酶Rho3相互作用,且Kin2偏爱与结合GTP的激活型Rho3相互作用。通过GST pull-down与双分子荧光互补,我们也检测到Kin2能够与Rho3在体内相互作用,且相互作用发生在极性生长位点,推测Rho3可能是一个作用于Kin2上游的调控分子,帮助解除Kin2分子C端对激酶结构域活性的抑制,对于激活Kin2的激酶活性有作用。我们发现提高Pho3Q74L的表达量能够促进过量表达Kin2造成的对septin组织的破坏,暗示着Rho3可能具有促进Kin2活性的功能,然而,我们在sec3-2、sec1-1和sec2-41等分泌功能缺陷的突变体中多拷贝表达Rho3后并没有发现Rho3对Kin2调控囊泡运输过程的功能具有促进作用,这一可能性是否存在尚需要其它实验来检验。我们还筛选到14-3-3蛋白Bmhl能够与Kin2相互作用,且Bmh1作用于Kin2的N端,我们推测Bmh1可能对于Kin2活性状态的维持有着重要的作用。Kin2同源蛋白在真菌中广泛存在,在一些致病性真菌,如新型隐球酵母、稻瘟病菌与禾谷镰刀菌中,Kin2同源蛋白是重要的致病因子之一,本课题研究成果对于揭示这些真菌的致病性机制具有重要的科学意义。
[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.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q936

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