|Budget Amount *help
¥14,800,000 (Direct Cost : ¥14,800,000)
Fiscal Year 2004 : ¥4,300,000 (Direct Cost : ¥4,300,000)
Fiscal Year 2003 : ¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 2002 : ¥5,500,000 (Direct Cost : ¥5,500,000)
In this research, based on our research background of membrane topogenesis and sorting, we have extensively examined whole topogenesis process of several typical membrane proteins into the endoplasmic reticulum membranes, including mechanism for all the transmembrane segments. The type I signal-anchor sequence, which translocates the N-terminal portion, mediates the translocation of N-domain much longer than had been assumed. For the translocation of the long N-domain, neither NTP's nor luminal hsp70 homologue, BiP, is required. Ribosome, however, plays critical function for the N-domain translocation. It is suggested to maintain the translocating chain at an appropriate position of the translocon. We also provided new evidences supporting our novel topogenic mode via which non-hydrophobic segment is forced to form transmembrane disposition by internal type I signal-anchor sequence. Furthermore, we extensively examined the mitochondrial targeting presequence which mediate the mitochondrial import of highly hydrophobic membrane proteins, ABC transporters and demonstrated the N-terminal 135 residues hydrophilic segment of ABC(B10) isoform is a membrane protein specific signal sequence that suppresses SRP-mediated ER targeting and mediate mitochondrial import. By the sequence the targeting mode of membrane proteins switches from co-translational ER translocation to the post-translational mitochondrial import. We also clarified topogeinc sequences of Tom22, mitochondrial outer membrane protein, and established experimental system for Tom40, by which Tom40 is over-expressed in E.coli, solubilized by denaturing reagent, purified, and renatured in vitro.