| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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