Insertion of Single-pass Transmembrane Proteins in the RER

The ER signal sequence acts as a start-transfer signal, initiating translocation of the polypeptide chain through the Sec61 channel on the ER membrane. This hydrophobic sequence is recognized by the translocon machinery and directs the growing protein into the channel. After the polypeptide begins crossing the lipid bilayer, the signal peptidase complex cleaves the signal sequence, releasing the N-terminal into the ER lumen and allowing continued protein synthesis.

A hydrophobic domain in the polypeptide chain cannot cross the lipid bilayer and acts as a stop-transfer signal when encountered by the Sec61 channel. The channel opens laterally at this point, releasing the hydrophobic domain directly into the lipid bilayer to form a transmembrane domain. This mechanism prevents further translocation and anchors the protein in the ER membrane while allowing ribosomal synthesis of the cytosolic domain to continue.

Type I proteins have a cleavable ER signal sequence and their N-terminal is positioned in the ER lumen with the C-terminal in the cytosol. Type II proteins lack a cleavable signal; instead, their transmembrane domain acts as a non-cleavable signal anchor sequence preceded by positively charged residues that prevent N-terminal translocation. This results in type II proteins having an inverted topology with the C-terminal in the lumen and N-terminal in the cytosol.

Positively charged residues positioned before the transmembrane domain prevent the N-terminal from slipping into the translocon, keeping it in the cytosol. In type II proteins, these residues precede the signal anchor sequence, resulting in cytosolic N-terminal orientation. In type III proteins, positive residues follow the signal anchor sequence, allowing N-terminal insertion into the translocon and leaving the C-terminal in the cytosol.

Transmembrane domains consist of 20-25 hydrophobic amino acids arranged in a helical secondary structure. These domains are stable enough to embed within the phospholipid interior of the membrane and are critical in determining the protein's overall topology. Their hydrophobic nature allows them to interact favorably with the nonpolar lipid environment while avoiding the aqueous cytoplasm and lumen.

The lateral gate of the Sec61 channel opens to allow hydrophobic domains to exit the translocon and embed directly into the lipid bilayer. The looped signal sequence uses this lateral gate to move out of the channel before being cleaved by signal peptidase. This mechanism enables efficient insertion of transmembrane domains without requiring the entire polypeptide to cross the membrane.

After translation terminates, the ribosome dissociates from the mRNA, leaving behind a type I signal transmembrane protein embedded in the ER membrane. The mature protein retains its characteristic topology with the N-terminal in the lumen and C-terminal in the cytosol. The insertion of multi-pass transmembrane proteins follows similar principles but involves multiple stop-transfer signals.