| Budget Amount *help |
¥15,660,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2007: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2006: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2005: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Research Abstract |
In this research, based on our research background of membrane topogenesis and sorting, we have extensively examined topogenesis process on the endoplasmic reticulum membranes and obtained the following progress. (1) The type I signal-anchor sequence, which translocates the N-terminal portion, can mediate the translocation of N-domain containing DHFR-domain. By using DHFR ligand, MTX, the N-terminal DHFR domain translocation can be regulated. For the translocation of the long N-domain, neither NTP's nor luminal hsp70 homologue, BiP, is required. Ribosome, however, plays a critical function for the N-domain translocation. The driving force for the N-domain at the initial stage is much larger than that of the latter stage. (2) The plant homologue of NHE gene family, NHX1 possesses the same membrane topology as that of animal, indicating the fundamental topology of the NHE gene family members. (3) In topogenesis of membrane proteins on the endoplasmic reticulum, the orientation of the hydrophobic transmembrane segment is influenced by the charge of the flanking amino acid residues. The timing of action of the charges during polypeptide elongation indicated that those function at the ribosome exit sites and the commitment of the hydrophobic segment to a particular orientation is influenced by far downstream parts of the polypeptide chain. (4) We developed novel regulatable experiment system for N-domain translocation. Two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. The remarkably flexible nature of the translocon was indicated.
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