Portions of the nascent chain are exposed to the lumen, the cytosol, or neither at different stages during the cotranslational integration of a protein into the ER membrane, as shown by compartment-specific collisional quenching of fluorophores incorporated into the polypeptide. The opening or closing of each end of the aqueous translocon pore is tightly controlled and occurs in a sequence that does not compromise the membrane's permeability barrier. Surprisingly, these structural changes at the membrane are effected by the transmembrane segment in the nascent protein from inside the ribosome. Thus, the ribosome, not the translocon, first recognizes the transmembrane segment and triggers long-range structural changes at the translocon that may be involved in shifting its function from translocation to integration.