The molecular mechanisms that accomplish co-translational protein translocation across and integration into the membrane of the endoplasmic reticulum (ER) at sites termed translocons are complex, in part because of the intrinsic difficulty of each process and in part because each process must be completed without compromising the permeability barrier established by the ER membrane. We have previously investigated protein trafficking from the point of view of the nascent protein chain by incorporating fluorescent dyes or photoreactive groups into the nascent chain as it is being made by the ribosome. By examining functional, fully-assembled, and intact translocation or integration intermediates with probes in the nascent chain, we have elucidated several important structural and mechanistic aspects of these processes. We now propose to extend our unique fluorescence and fluorescence resonance energy transfer (FRET) investigations of trafficking by addressing questions that include: Which domains and functions of BiP are required for the BiP-dependent closing of the pore and for the opening of the pore when BiP is released? Does the ribosome induce all nascent chain transmembrane (TM) sequences to fold into an alpha-helix (or nearly so) far inside the exit tunnel? Do TM sequences oriented in the bilayer in opposite directions regulate ribosome-translocon-BiP interactions differently, with N(lum)-C(cyto) TM sequences initiating BiP-mediated closure of the lumenal end of the pore via a long transmembrane signal transduction pathway and N(cyto)-C(lum) TM sequences alternately triggering the closure of the cytosolic end of the pore by the ribosome? When do the TM sequences of a multi-spanning membrane protein (MSMP) begin to assemble into their native structure? We will also use a novel variation of the photocrosslinking approach to determine whether the TM segments in a MSMP move through the translocon and into the bilayer singly, in pairs, or all at once. In addition, we will create a reconstituted system with fluorescent-labeled substrates of the dislocation or ER-associated degradation (ERAD) pathway to characterize and quantify various aspects of the retrotranslocation of misfolded and unassembled proteins from the ER lumen to the cytosol, and thereby assess the role of various cytosolic, lumenal, and membrane proteins on ERAD targeting and retrotranslocation. This basic research will elucidate aspects of protein sorting at the molecular level in normal cells and provide a context for identifying irregularities in abnormal cells.