Structure and function of the novel factor involved in protein integration into the cytoplasmic membrane of E.coli

参与蛋白质整合到大肠杆菌细胞质膜中的新因子的结构和功能

• 批准号: 17570090
• 负责人: NISHIYAMA Ken-ichi
• 金额: $ 2.3万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570090/
• 关键词: integration of membrane protein; SRP; secYEG; protein secretion; E.coli; MtlA; reconstitution

Molecular mechanisms underlying membrane protein integrations in E.coli were investigated by means of the reconstitution system. A novel factor that is involved in protein integrations was identified, and the relationship between its structure and function was studied. We found that MtlA, of which integration requires SecYEG strictly, is spontaneously integrated into liposomes composed of phospholipids. This spontaneous integration was blocked by diacylglycerol if contained in the liposomes at the physiological concentration. Diacylglycerol blocked the spontaneous integrations not only of MtlA but also of M13 procoat protein which has been thought to be integrated spontaneously since >20 years. Thus, t was suggested that the cell has an ability to block the disordered integrations, and that diacylglycerol plays a central role in this event. In the presence of diacylglycerol, we could develop the reconstitution system, demonstrating that in addition to SecYEG a novel factor was essentia … More l for MtlA integration. The factor was 〜8kDa on an SDS-gel. Although the integration activity was lost upon proteinase digestion, the most part of the factor was turned out to be lipid and glycan moieties. Moreover, the biosynthesis of the factor was affected by the mutations involved in lipopolysaccharides (LPS) biosynthesis, indicating that the factor is a lipid A-derivative possessing a peptide moiety.We also found that the cytosolic factors other than integration factors such as SecA/SecB and Ffh/FtsY are dispensable for membrane protein integrations. Regarding the expression of the Sec factors, we demonstrated that the rpmJ gene encoding ribosomal protein, L36, is involved in the expression of the immediate upstream gene, secY, and that the expression of the secG gene is coupled with the immediate downstream gene, leuU, encoding a tRNA for leucine. These observations strongly suggest the tight relationship between protein synthesis and the subsequent protein translocation/integration. Less

通过重构系统研究了大肠杆菌中膜蛋白整合的分子机制。鉴定出参与蛋白质整合的新因素，并研究了其结构和功能之间的关系。我们发现，MTLA严格要求其进行整合，它被赞助为由磷脂组成的脂质体。如果在物理浓度下脂质体中含有二酰基甘油，则将这种赞助的整合被阻塞。二酰基甘油不仅阻止了赞助商的自发性整合，而且还阻止了M13 procoat蛋白的蛋白质，这被认为是自20年以来自发综合的。这被认为，该细胞具有阻断无序整合的能力，并且二酰基甘油在此事件中起着核心作用。在存在二酰基甘油的情况下，我们可以开发重建系统，表明除了secyeg外，新的因素是必不可少的……更多的MTLA整合因子。 SDS-凝胶上的因素约为8KDA。尽管在蛋白酶消化时损失了整合活性，但该因子的大部分是脂质和聚糖部分。此外，该因子的生物合成受到脂多糖（LPS）生物合成的突变的影响，表明该因子是具有肽部分具有肽的脂质A源性。我们还发现，除了seca/seca/seca/secb/ffh/ffh/ffh/ffh/ffh/ftsy的胞质因子以外的细胞质因子以符合效率的效果。关于SEC因子的表达，我们证明了编码核糖体蛋白L36的RPMJ基因参与直接上游基因Secy的表达，并且SECG基因的表达与直接下下游基因Leuu耦合，以编码Leucine的TRNA。这些观察结果强烈表明蛋白质合成与随后的蛋白质易位/整合之间的紧密关系。较少的

项目成果

大腸菌における膜内在性タンパク質の膜挿入機構

大肠杆菌整合膜蛋白的膜插入机制

• 发表时间: 2007
• 期刊: 生化学 79
• 作者: Kagoshima H;Sawa H;Mitani S;Burglin TR;Shigesada K;Kohara Y;西山賢一
• 通讯作者: 西山賢一

Genes coding for SecG and Leu2-tRNA form an operon to give a unusual RNA comprising mRNA and a tRNA precursor

编码 SecG 和 Leu2-tRNA 的基因形成操纵子，产生包含 mRNA 和 tRNA 前体的不寻常 RNA

• 发表时间: 2005
• 期刊: Biochim.Biophys.Acta 1729
• 作者: Nishiyama;K.
• 通讯作者: K.

Genes coding for SecG and Leu2-tRNA form an operon to give an unusual RNA comprising mRNA and a tRNA precursor

编码 SecG 和 Leu2-tRNA 的基因形成操纵子，产生包含 mRNA 和 tRNA 前体的不寻常 RNA

• 发表时间: 2005
• 期刊: Biochim.Biophys.Acta 1729
• 作者: Nishiyama;K.
• 通讯作者: K.

Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

• DOI: 10.1021/bp049553u
• 发表时间: 2005-07-01
• 期刊: BIOTECHNOLOGY PROGRESS
• 影响因子: 2.9
• 作者: Kuruma, Y;Nishiyama, K;Ueda, T
• 通讯作者: Ueda, T

Disruption of rpmJ encoding ribosomal protein L36 decreases the expression of secY upstream of the spc operon and inhibits protein translocation in Escherichia coli

• DOI: 10.1271/bbb.69.1595
• 发表时间: 2005-08-01
• 期刊: BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
• 影响因子: 1.6
• 作者: Ikegami, A;Nishiyama, K;Tokuda, H
• 通讯作者: Tokuda, H