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大肠杆菌膜蛋白整合的分子机制。鉴定了一种新的参与蛋白质整合的因子，并对其结构与功能的关系进行了研究。我们发现，MtlA，其整合需要SecYEG严格，自发地整合到脂质体组成的磷脂。如果脂质体中含有生理浓度的甘油二酯，则这种自发整合被阻断。二酰基甘油不仅阻断MtlA的自发整合，而且阻断M13前外壳蛋白的自发整合，M13前外壳蛋白自>20年以来一直被认为是自发整合的。因此，这表明细胞具有阻断无序整合的能力，并且二酰基甘油在该事件中起着核心作用。在甘油二酯的存在下，我们可以开发重构系统，证明除了SecYEG之外，还需要一种新的因子 ...更多信息 l为MtlA整合。SDS-凝胶电泳测得该因子分子量为108 kDa。虽然整合活性在蛋白酶消化后丧失，但大部分因子被证明是脂质和聚糖部分。此外，该因子的生物合成受到脂多糖（LPS）生物合成中所涉及的突变的影响，表明该因子是具有肽部分的脂质A衍生物。我们还发现，除了整合因子SecA/SecB和Ffh/FtsY之外，胞质因子也参与膜蛋白的整合。关于Sec因子的表达，我们证明了编码核糖体蛋白L36的rpmJ基因参与了直接上游基因secY的表达，并且secG基因的表达与直接下游基因leuU（编码亮氨酸的tRNA）相结合。这些观察结果强烈表明蛋白质合成和随后的蛋白质易位/整合之间的紧密关系。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