Analysis of an actin-related protein localized in nuclei

位于细胞核中的肌动蛋白相关蛋白的分析

基本信息

批准号：
08044188
负责人：
HARATA Masahiko
金额：
$ 2.75万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Recently various actin-related proteins, which show moderate similarity to conventional actins, have been reported, suggesting that actin family is much more diverse than it had been thought. ACT3 in Saccharomyces cerevisiae codes an actin-related protein and is essential for viability. Act3p is an novel actin-family protein in terms of its nuclear localization. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3p. On the other hand, Act3p contains two unique hydrophilic segments compared with other actin family proteins. One of the unique segments contains a putative nuclear-targeting signal. The other is abundant in charged amino acids and predicted to form a loop-like structure protruded from surface.Little is known about function of Act3p in nucleus, however, we got some results suggesting that Act3p was associated with chromatin. For instance, Act3p is released from isolated nuclei by treatment with salt or by digestion of chromatin with DNaseI.In addition, it was shown that point-mutations of ACT3 cause epigenetic effects on both chromatin structure and transcription of some genes in yeast.It was thought that the unique segments of Act3p would be involved in its distinct function from other actin-family Proteins and we tried to identify proteins which interact with the segments. In the course of the research, we perfbrmed far-western blotting with a labeled peptide containing one of the segments and two-hybrid analysis. As a result, it was shown that the peptide bound mainly to core histones in vitro. In addition, protein-complex (es) containing both Act3p and core histones was isolated from nuclear extract using histidine-tagged Act3p and an anti-Act3p antibody, suggesting that Act3p interacts with core histones in vivo.These findings show that Act3p is involved in transcriptional regulation through the change and/or maintenance of chromatin structure.

最近，已经报道了与常规肌动蛋白相似的各种与肌动蛋白相关的蛋白质，这表明肌动蛋白家族比以前想象的要多样化得多。酿酒酵母中的ACT3代码是肌动蛋白相关的蛋白质，对于生存力至关重要。就其核定位而言，ACT3P是一种新型的肌动蛋白蛋白质。根据序列比较，传统肌动蛋白的基础核心结构可以在ACT3P中得到保守。另一方面，ACT3P与其他肌动蛋白家族蛋白相比包含两个独特的亲水性片段。独特的部分之一包含推定的核定靶向信号。另一个在带电的氨基酸中很丰富，预计会形成从表面突出的环状结构。例如，ACT3P通过用盐处理或通过DNAsei进行染色质而从孤立的核中释放出来。此外，证明ACT3的点突变会对酵母中的染色质结构和某些基因的转录引起表观遗传作用。认为ACT3P的独特片段将与其他肌动蛋白蛋白的独特功能有关，我们试图鉴定与片段相互作用的蛋白质。在研究过程中，我们用包含一个片段和两种杂交分析的标记肽进行了详尽的远面印迹。结果，结果表明，肽主要结合在体外与核心组蛋白结合。此外，使用组氨酸标记的ACT3P和一种抗ACT3P抗体从核提取物中分离出含有ACT3P和核心组蛋白的蛋白质复合物（ES），这表明ACT3P在Vivo中与核心组蛋白相互作用。这些发现表明ACT3P参与了ACT3P参与通过更改和/或维护染色质结构的转录调节。