The rough ER membrane synthesizes, assembles, and embeds transmembrane proteins in diverse topologies. These proteins function as transporters or channels and can remain in the ER membrane or are sent to the Golgi complex, lysosome, and cell membrane.

The multipass transmembrane proteins are the type IV integral membrane proteins with multiple topogenic sequences determining their spatial arrangement in the ER membrane. Nearly all multipass proteins lack a cleavable signal sequence and use their first hydrophobic or transmembrane domain as the ER signal sequence. The positioning of positive residues before or after the first transmembrane domain determines the orientation of the N terminal of the protein in the cytosol or lumen, respectively.

The signal recognition particle (SRP) and its receptor (SR) are required to initiate the translocation of the first transmembrane domain of a multipass membrane protein through the ER membrane. The threading of subsequent transmembrane domains is independent of the SRP-SR complex. It is managed by the ribosome-translocon assembly and is primarily dependent on the hydrophobicity of the translated domain. The N-terminal of multipass proteins like glucose transporters and Sec61 lies in the cytosol, while that of G protein-coupled receptors are placed in the ER lumen.

The insertion of single-pass transmembrane proteins is understood better as compared to the multipass transmembrane proteins. The multipass proteins have complex biophysical and topological features affected by the length and hydrophobic profile of their transmembrane domains, the distance between consecutive transmembrane domains, and the length of the extra-membrane loops. The Sec61 translocon-ribosome assembly forms the core of protein translocation of membrane proteins. However, it is an overly simplified model to explain the incorporation of multipass proteins. Different accessory complexes like TRAP and TRAM interact with the Sec61 translocon and assist in accurate folding, insertion, and assembly of multipass transmembrane proteins.