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

利用重组系统研究了在大肠杆菌中整合膜蛋白的分子机制。确定了一个参与蛋白质整合的新因子，并研究了其结构与功能之间的关系。我们发现，MtlA的整合严格要求SecYEG，它自发地整合到由磷脂组成的脂质体中。如果脂质体中含有生理浓度的二酰甘油，这种自发的整合就会被阻断。二酰甘油不仅阻止了MtlA的自发整合，也阻止了M13蛋白的自发整合，而M13蛋白自20年来一直被认为是自发整合的。因此，有人认为细胞具有阻止无序整合的能力，而二酰甘油在这一事件中起着核心作用。在双甘油存在的情况下，我们可以发展重建系统，证明除了SecYEG之外，还有一个新的因子是Essentia…更多关于MTLA整合的L。在十二烷基硫酸钠凝胶上约为8 kDa。虽然酶消化后失去了整合活性，但该因子的大部分是脂类和糖类。此外，该因子的生物合成受到脂多糖生物合成所涉及的突变的影响，表明该因子是一种含有多肽的脂类A-衍生物。此外，我们还发现，除了整合因子SecA/SecB和FFH/FtsY之外，细胞质因子对于膜蛋白的整合是必不可少的。关于SEC因子的表达，我们证明了编码核糖体蛋白的rpmJ基因L36参与了立即上游基因Secy的表达，并且SecG基因的表达与立即下游基因LeuU偶联，后者编码亮氨酸的tRNA。这些观察结果有力地表明了蛋白质合成和随后的蛋白质转位/整合之间的密切关系。较少

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