The molecular mechanisms that accomplish protein targeting to, 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 investigated these processes 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 fully-assembled translocation intermediates with fluorescent probes in the nascent chain, we showed that secretory proteins are co- translationally translocated across the ER membrane through an aqueous pore that completely spans the bilayer. We have since discovered that the translocon undergoes very unexpected and dramatic changes in structure and composition in order to ensure that one end or the other of the aqueous translocon pore is closed at each stage of translocation and integration. We propose to extend our unique fluorescence investigations of translocon structure and function by addressing questions that include: What transiently seals the lumenal end of the pore during integration? Does the lateral movement of a transmembrane (TM) segment into the bilayer involve specific protein-protein interactions with translocon components? Which ER proteins form the cytosolic seal with the ribosome, and the lumenal seal with BiP? What translocon structural changes accompany the decrease in pore diameter from 40-60 Angstrom units in a functioning translocon to 9-15 Angstrom units in a ribosome-free translocon? We will also use fluorescence spectroscopy and resonance energy transfer to quantify, directly and at equilibrium, SRP affinity for ribosome-bound signal sequences and the dependence of this affinity on GTP and the length of the nascent chain. In addition, we will determine whether the TM segments in a multi- spanning membrane protein move through the translocon and into the bilayer singly, in pairs, or all at once. 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.