Protein translocation across the ER membrane
Protein translocation is the process by which peptides are transported across a membrane bilayer. Translocation of proteins across the membrane of the membrane of the endoplasmic reticulum (ER) is know to occur in one of two ways: cotranslationally, in which translocation is concurrent with peptide synthesis by the ribosome, or posttranslationally, in which the protein is first synthesized in the cytosol and later is transported into the ER. Both means of translocation are mediated by the same protein channel, known as Sec61 in eukaryotes and SecY in prokaryotes and archaea.
Proteins that are targeted for translocation across the ER membrane have a distinctive amino-terminal signal sequence (shown in yellow in the animation) which is recognized by the signal recognition particle (SRP). The SRP in eukaryotes is a large ribonucleoprotein which, when bound to the ribosome and the signal sequence of the nascent peptide, is able to arrest protein translation by blocking tRNA entry.
The ribosome is targeted to the ER membrane through a series of interactions, starting with the binding of the SRP by the SRP receptor. The signal sequence of the nascent peptide chain is then transferred to the protein channel, Sec61. The binding of SRP to its receptor causes the SRP to dissociate from the ribosome, and the SRP and SRP receptor also dissociate from each other following GTP hydrolysis. As the SRP and SRP receptor dissociate from the ribsome, the ribosome is able to bind directly Sec61.
The Sec61 translocation channel (known as SecY in prokaryotes) is a highly conserved heterotrimeric complex composed of α-, β- and γ-subunits. The pore of the channel, formed by the α-subunit, is blocked by a short helical segment which is thought to become unstructured during the beginning of protein translocation, allowing the peptide to pass through the channel. As shown in the animation, the signal sequence of the nascent peptide intercalates into the walls of the channel, through a side opening known as the lateral gate. During translocation, the signal sequence is cleaved by a signal peptide peptidase, freeing the amino terminus of the growing peptide.
Posttranslational translocation in eukaryotes
During cotranslational translocation, the ribosome provides the motive power that pushes the growing peptide into the ER lumen. During posttranslational translocation, additional proteins are necessary to ensure that the peptide moves unidirectionally into the ER membrane. Eukaryotes and prokaryotes have evolved different mechanisms to ensure the successful translocation of synthesized peptides.
In eukaryotes, posttranslational translocation requires the Sec62/Sec63 complex (shown in green in the animation) and the chaperone protein BiP (shown in purple/blue).
BiP is a member of the Hsp70 family of ATPases, a group which is characterized as having an N-terminal nucleotide-binding domain (NBD), and a C-terminal substrate-binding domain (SBD) which binds to peptides. The nucleotide binding state of the NBD determines whether the SBD can bding to a substrate peptide. While the NBD is bound to ATP, the SBD is in an open state, allowing for peptide release, while in the ADP state, the SBD is closed and peptide-bound. In the animation, the ATP-bound NBD is shown in purple, and the ADP-bound state is shown in blue.
The primary role of the membrane protein complex Sec62/Sec63 is to activate the ATPase activity of BiP via a J-domain located on the lumen-facing portion of Sec63. The SBD of BiP binds non-specifically to the peptide as it enters the ER lumen, and keeps the peptide from sliding backwards in a ratchet-type mechanism.
Posttranslational translocation in prokaryotes
In prokaryotes, translated peptides are actively pushed through the SecY channel by a protein called SecA. SecA is composed of a nucleotide-binding domain (medium green), a polypeptide crosslinking domain (dark green), and helical wing and scaffold domains (light green).
During translocation, a region of the helical scaffold domain forms a two-finger helix which inserts into the cytoplasmic side of the SecY channel, thereby pushing the translocating peptide through. A tyrosine found on the tip of the two-finger helix plays a critical role in translocation, and is thought to make direct contact with the translocating peptide.
The polypeptide crosslinking domain (PPXD) forms a clamp which is thought to open as the translocating peptide is being pushed into the SecY channel by the two-finger helix, and close as the two-finger helix resets to its "up" position.
The conformational changes of SecA are powered by its nuclease activity, with one ATP being hydrolyzed during each cycle.
Please note that animations and illustrations from this website are licensed under a Creative Commons License, and may be freely downloaded for non-commercial uses with proper attribution. See link at bottom of page for more information.
[download quicktime movie (20.8 MB)]
this movie can also be downloaded as separate segments:
Part I: Docking
[download quicktime movie (13.0 MB)]
Part II: Sec61 structure
[download quicktime movie (6.8 MB)]
Part III: Peptide elongation and release
[download quicktime movie (3.9 MB)]
posttranslational translocation in eukaryotes
[download quicktime movie (13.0 MB)]
posttranslational translocation in prokaryotes
[download quicktime movie (14.2 MB)]
Crystal structures used as references are:
secY: 1RHZ [van den Berg, et al., 2003]
SRP: 1RY1 [Halic, et al., 2003], 2J37 [Halic, et al., 2006]
SRP receptor: 2GO5 [Halic, et al., 2006], 1RJ9 [Egea, et al., 2003]
ribosome: 1S1H (40s subunit), 1S1I (60s subnit) [Spahn, et al., 2004]
secA: 1M6N, 3DIN [Zimmer, et al., 2008]
Many thanks to Tom Rapoport, Mario Halic and Jochen Zimmer (Harvard Medical School) for collaborating on this project.This work by Janet Iwasa is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.