Investigating cotranslational integration of a multi-spanning membrane protein into the endoplasmic reticulum membrane

Most membrane proteins in eukaryotic cells are co-translationally integrated into the endoplasmic reticulum (ER) membrane at aqueous pores termed translocons. During multi-spanning membrane protein (MSMP) integration, the nascent polypeptide is threaded into the translocon pore where each successive transmembrane segment (TMS) is moved laterally through the translocon into the bilayer. The hydrophilic polypeptide segments on each side of the TMS are alternately directed into either the aqueous cytosol or the aqueous ER lumen. How is the ER membrane permeability barrier maintained during this process? For a single-spanning signal-cleaved membrane protein, nascent chain movement into the lumen occurs while an ion-tight ribosome-translocon junction prevents ion flow through the translocon pore. Prior to opening this junction to allow nascent chain movement into the cytosol, BiP (Hsp70 binding protein) effects closure at the lumenal end of the pore to maintain the membrane permeability barrier. To determine whether the ribosome and BiP alternately mediate pore closure during the integration of a MSMP, integration intermediates with nascent chains of different lengths were prepared with a fluorescent probe positioned in the nascent chain far inside the ribosomal tunnel. Nascent chain exposure to the cytosol or lumen was then detected by the collisional quenching of the probe by iodide ions located on either the cytosolic or lumenal side of the membrane. While the first TMS through the tunnel caused the ribosome-translocon junction to open, the second TMS elicited both the closure of this junction and the opening of the lumenal end of the pore. Movement of a third TMS through the tunnel caused the ribosome-translocon junction to re-open after closure of the lumenal end. Pore opening and closing occurred after each TMS was 4-7 residues from the peptidyltransferase center, irrespective of TMS location in the nascent chain. The ribosome treated all TMSs in the same manner, regardless of their individual sequence or their native orientation. The ER membrane permeability barrier is maintained by ribosome-translocon interactions during cotranslational MSMP integration