拟南芥CSN1对COP1细胞内定位的影响以及CSN1生物学功能的进一步探讨

【作者】 李雯俊；

【导师】 曹凯鸣； 王喜萍；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 COP9信号复合体(CSN)是一个保守的蛋白复合体,由8个不同的亚基组成,依次命名为CSN1-CSN8。CSN复合体最初是在对拟南芥光形态发生的遗传学研究中被发现的,csn突变体幼苗都呈现组成型光形态建成的表型。CSN. COP1以及COP10-DDB1-DET1 (CDD)复合体都是调节植物光反应和发育的关键因子。COP1受到光信号影响会改变其在细胞核与细胞质中的分布比例。早期拟南芥遗传学研究发现COP1的细胞核定位需要CSN复合体的存在,但CSN复合体影响COP1的细胞内定位的分子机制还没有报道。为了探讨COP1在细胞内定位的分子机制,利用洋葱表皮细胞瞬间表达系统,我们发现共表达的CSN1能够促进GUS-COP1的细胞核定位,且CSN1的N-末端是促进GUS-COP1细胞核定位的关键。同时利用拟南芥csn1突变体,证实CSN1的N-末端是COP1实现细胞核定位不可缺少的蛋白片段。进而利用酵母双杂交系统检测发现CSN1通过其N-末端结构域直接和COP1的coiled-coil结构域发生相互作用,并且通过免疫共沉淀实验证实COP1和CSN复合体在拟南芥体内存在相互作用。此外,CSN1对COP1细胞核定位的促进作用需要COP1自身细胞核定位信号的参与,同时也与COP1的coiled-coil吉构域有关。COP1的coiled-coil结构域不仅是CSN1的结合区,还包含了COP1细胞质定位信号的一部分。这些发现使我们CSN复合体影响COP1的细胞定位的机制有了进一步的了解,为今后深入研究CSN复合体调控植物光形态发生的分子机制提供了一定的依据。CSN复合体最初被定义为拟南芥光形态发生的抑制因子,随着研究的深入,人们发现CSN复合体参与真核生物的各种生长发育过程以及细胞周期等细胞变化进程。CSN1亚基的C-末端包含一个PCI结构域,该结构域负责蛋白亚基与业基的相互作用以及CSN复合体的形成。CSN1的N-末端不参与CSN复合体的形成,但是对CSN复合体的功能起着重要作用。为了深入了解CSN复合体如何通过CSN1调控植物的生长发育等过程,本研究利用酵母双杂交文库筛选技术寻找CSN1的N-末端相互作用蛋白,获得了5个候选蛋白,分别是PAD2、TSA1、eIF-2家族蛋白、核糖体60S亚基蛋白L35以及一个含C2结构域的蛋白,并对其中的PAD2、TSA1的相关功能进行了初步研究。PAD2是26S蛋白酶体的核心组分20S蛋白酶体的α亚基之一。已有证据揭示拟南芥中CSN复合体、26S蛋白酶体和SCF型E3连接酶能够相互结合形成更大的复合体,而且拟定南芥SCF型E3可以通过Skp1亚基SnRK-蛋白激酶的直接相互作用锚定到PAD1上。但目前关于CSN复合体通过何种方式参与其中还不是很清楚,通过酵母双杂交文库筛选,我们发现PAD2的C-末端片段与CSN1的N-末端片段存在相互作用。进一步研究发现与PAD2高度同源的蛋白PAD1的C-末端也能和CSNl的N-末端发生相互作用。此外,在酵母双杂交系统中,我们发现PAD1的C-末端与COP1也存存相互作用,但是PAD2的C-末端与COP1没有相互作用。由此我们推测CSN复合体可以通过CSN1的N端与26S蛋白酶体发生相互作用,起到维持更大复合体的稳定的作用或者也可能调节26S蛋白酶体的相关活性,而且COP1作为另一类E3连接酶也可能通过PAD1来参加蛋白降解过程,相关机制尚需进一步的研究。TSA1是一种钙调结合蛋白相关蛋白,可以与TSK相互作用,并且可能参与细胞周期过程的调控。通过酵母双杂交文库筛选,我们发现TSAl的C-末端与CSNl的N-末端片段存在相互作用。酵母双杂交分析证实TSA1全长和CSN1的N-末端存在相互作用。为了研究TSA1的生物学功能,我们获得了TSAl的T-DNA插入突变体。检测发现该突变体的T-DNA插入位点在TSA1基因ATG上游103个碱基处。半定量RT-PCR检测发现突变体中TSA1的转录水平比野生中TSA1的转录水平低。在暗培养条件下,tsa1突变体幼苗的下胚轴与野生型相比略短,但在光培养条件下,则没有明显变化。这一实验结果表明TSA1可能在光形态建成过程中承担某种功能,进一步的研究尚在进行之中。更多还原

【Abstract】 The C0P9 signalosome (CSN) complex is a conserved protein complex, typically consisting of eight subunits designated CSN1-CSN8. CSN complex is originally identified based on mutants with a constitutive photomorphogenic (cop) phenotype in Arabidopsis thaliana seedlings.CSN, COP1 and C0P10-DDB1-DET1 (CDD) complex component are key regulators of plant light responses and development. COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicates that nuclear localization of COP1 requires CSN. However the mechanism underlying the functional relationship between COP1 and CSN is unknown.In this report, we report that, in onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. We also provide genetic evidence that CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyls cells. Further, we demonstrate that CSN1, via its N-terminal domain (NTD), directly interacts with COP1 at the coiled-coil domain in yeast two hybrid system, and confirm that COP1 weakly associates with CSN in vivo. Moreover, CSN1-induced COP1 nuclear localization requires COP1’s own nuclear-localization sequences as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. These findings provide a mechanistic insight into the interrelationship between COP1 and the CSN complex. Our findings set a foundation for further investigations into the mechanism of COP1 subcellular localization in plants.CSN complex is initially defined as a repressor of photomorphogenesis in Arabidopsis, and it has now been found to participate in diverse cellular and developmental processes in various eukaryotic organisms. CSN1 contains a large PCI domain located at the C-terminal half of the protein, which is necessary for subunit-subunit interaction and complex assembly. The NTD of CSN1, on the other hand, does not have a structural role, but is involved in a critical function of CSN that is not yet understood molecularly.To get more information about the functional contribution of CSNl-NTD in plant development, yeast two-hybrid screening is used to identify proteins able to interact with the NTD of CSNl. At last, five different genes are identified by sequencing the positive clones. The proteins of these genes are PAD2, TSA1, eIF-2 family protein,60S ribosomal protein L35 and C2 domain-containing protein. We focus on the study of the functions of PAD2, TSA1 with CSN1-NTD.PAD2 is one of the 20S proteasome alph subunits. By yeast two-hybrid screening, we got the NTD truncated version of PAD2. In Arabidopsis, PAD2 has a homology, named PAD1. We find that the C-terminal domain (CTD) of PAD1 can also interact with CSN1-NTD in yeast two-hybrid system. Moreover, the interaction between COP1 and the CTD of PAD1, but not the CTD of PAD2, is detected by yeast two-hybrid assay.TSA1 is a caldesmon-related protein, and has Ca2+-binding activity. By yeast two-hybrid screening, we got the NTD truncated version of TSA1. The interaction between full length TSA1 and CSN1-NTD is confirmed by yeast two-hybrid assay. To study the biological function of TSA1, T-DNA insertion mutant is obtained from the SALK T-DNA collection. In the line, T-DNA insertion is 103bp before the ATG of AtTSAl. The expression level of AtTSA1 in the mutant is examined by half-quantitative RT-PCR, which shows lower TSA1 transcript in the mutant than in the wild type. In normal light growth conditions, tsal mutants do not exhibit any detectable abnormality throughout the whole life span. While in the dark, the mutants exhibit slightly decreased hypocotyls, compared with the wild type.Further studies are required to del ineate the functional relationship between CSNl and these interaction proteins.更多还原