Tail-anchoring of Proteins in the ER Membrane

Tail-anchored (TA) proteins are a specialized class of membrane proteins with a single hydrophobic transmembrane domain at their C-terminal end, representing 3-5% of eukaryotic membrane proteins. Unlike typical membrane proteins, their transmembrane domain is positioned approximately 30 amino acids upstream from the C-terminus, allowing it to serve as both an anchor and an ER signal sequence. Examples include Sec61β and cytochrome b5.

The signal recognition particle (SRP) cannot guide TA proteins to the ER because their transmembrane domain is located at the C-terminal end rather than near the N-terminus. Since TA proteins are synthesized completely before their signal sequence emerges from the ribosome, cotranslational insertion is impossible. Instead, TA proteins require post-translational translocation of proteins to the ER using specialized pathways like the GET system.

The GET pathway begins when Sgt2, a protein chaperone, recognizes the C-terminal hydrophobic domain of a newly synthesized TA protein. Sgt2 forms a pre-targeting complex with Get4 and Get5 to shield the hydrophobic domain. This complex then interacts with ATP-bound Get3 ATPase, which carries the TA protein to the Get1/Get2 heterotetrameric complex on the ER membrane. ATP hydrolysis by Get3 triggers conformational change, enabling TA protein insertion into the membrane.

Get3 is a homodimeric ATPase that alternates between two ATP-dependent conformations to transport TA protein cargo to the ER membrane. In its ATP-bound state, Get3 accepts the TA protein from the pre-targeting complex and guides it toward the Get1/Get2 insertase. When ATP is hydrolyzed, Get3 switches to an open conformation, facilitating TA protein transfer to the membrane complex. After insertion, Get3 is recycled back to the cytosol.

TA proteins with highly hydrophobic domains use an SRP-dependent pathway paired with the Sec61 channel for lateral membrane insertion. Proteins with low-hydrophobicity TA domains employ chaperone-mediated membrane insertion using HSP70/40 molecules. The SND-targeting pathway, comprising SND1, SND2, and SND3 proteins, represents a more recently discovered alternative. These pathways demonstrate that the canonical GET pathway is not the exclusive mode of TA protein insertion in yeast and mammalian cells.

After successful insertion via the GET pathway, a tail-anchored protein has a free N-terminal domain extending into the cytosol while its C-terminal transmembrane domain is embedded within the ER membrane. This orientation is achieved because the TA protein's C-terminal hydrophobic domain serves as the membrane anchor, positioning the bulk of the protein on the cytoplasmic side of the ER membrane.

The Get1/Get2 heterotetrameric complex acts as the membrane insertase for TA proteins. The cytosolic domain of Get2 first captures the Get3-TA protein complex, and this interaction is further stabilized by the Get1 cytosolic domain. Once the Get3-TA protein complex is secured at the membrane, ATP hydrolysis by Get3 triggers the conformational change necessary for the Get1/Get2 complex to insert the TA protein into the ER membrane lipid bilayer.