The application seeks to understand the mechanism of posttranslational translocation across the membrane of the endoplasmic reticulum of Saccharomyces cerevisiae. Specific Aim 1 addresses the mechanism of targeting of substrates. The presence and importance of cytosolic chaperones or targeting molecules will be assessed. The second Specific Aim will determine the role of the signal sequence on the gating of the translocating channel. It will test the hypothesis that the signal sequence displaces a component of the translocon, and as a result, opens the translocation channel.. Specific Aim 3 is to determine the stoicheometry of the components of the translocon. Estimations of 3 or 4 Sec61p complexes comprising the translocation channel have been made based on electron micrographic techniques, and this will be tested biochemically. A role of Sec62p in catalyzing assembly will be tested. Finally, Specific Aim 4 seeks to understand the role of luminal Hsp70 in the translocation process. It will test the hypothesis that this protein works as a molecular ratchet rather than a machine that "pulls" the protein through the membrane.