Mechanism of organelle targeting and topogenesis of membrane proteins

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The primary objective of this study is to establish a reliable system for prediction of membrane topology, intracellular localization, and function of all membrane proteins encoded in the genome through elucidation of principal mechanisms for the topogenic process and the localization. On the basis of topogenic mechanisms on the endoplasmic reticulum, we have extensively examined the membrane integration of all the transmembrane segment of typical multispanning membrane proteins. We demonstrated that the type I signal anchor sequence can translocate N-terminal domain which is longer than we have expected. For the long N-domain translocation, no nucleotide triphosphates, no lumenal hsp70-homologue (BiP) are required. Ribosome plays an essential role for the translocation even after membrane targeting. The driving force for the N-domain translocation at the initial step is larger than that for the following continuous translocation. Furthermore, we systematically examined the signal sequences for targeting of membrane proteins to mitochondria and peroxisome. The long mitochondrial targeting presequence suppresses the co-translational targeting of hydrophobic membrane proteins to the endoplasmic reticulum mediated by signal recognition particle and translocon, and mediates the post-translational targeting to mitochondria. We elucidated targeting signals of mitochondrial outer membrane proteins, TOM22 and TOM20. To address the molecular mechanism of recognition of mitochondrial signal sequence, we established the system for expression, purification, and refolding of mitochondrial outer membrane protein translocation channel, TOM40.