Translocation requires insertion into the translocon and (sometimes) a ratchet in the ER

KEY CONCEPTS:

• The ribosome, SRP, and SRP receptor are sufficient to insert a nascent protein into a translocon.
• Proteins that are inserted post-translationally require additional components in the cytosol and Bip in the ER.
• Bip is a ratchet that prevents a protein from slipping backward.

The translocon and the SRP receptor are the basic components required for co-translational translocation. When the Sec61 complex is incorporated into artificial membranes together with the SRP receptor, it can support translocation of some nascent proteins. Other nascent proteins require the presence of an additional component, TRAM, which is a major protein that becomes crosslinked to a translocating nascent chain. TRAM stimulates the translocation of all proteins.

The components of the translocon and their functions are summarized in Figure 8.28. The simplicity of this system makes several important points. We visualize Sec61 as forming the channel and also as interacting with the ribosome. The initial targeting is made when the SRP recognizes the signal sequence as the newly synthesized protein begins to emerge from the ribosome. The SRP binds to the SRP receptor, and the signal sequence is transferred to the translocon. When the signal sequence enters the translocon, the ribosome attaches to Sec61, forming a seal so that the pore is not exposed to the cytosol. Cleavage of the signal peptide does not occur in this system, and therefore cannot be necessary for translocation per se. In this system, components on the lumenal side of the membrane are not needed for translocation.

Of course, the efficiency of the in vitro system is relatively low. Additional components could be required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process (Gorlich and Rapoport, 1993; for review see Walter and Lingappa, 1986; Rapoport, Jungnickel, and Kutay, 1996).

A more complex apparatus is required in certain cases in which a protein is inserted into a membrane post-translationally. The same Sec61 complex forms the channel, but four other Sec proteins are also required, and in addition the chaperone BiP (a member of the Hsp70 class) and a supply of ATP are required on the lumenal side of the membrane. Figure 8.29 shows that BiP behaves as ratchet (Matlack et al., 1999). In the absence of BiP, Brownian motion allows the protein to slip back into the cytosol. But BiP grabs the protein as it exits the pore into the endoplasmic reticulum. This stops the protein from moving backward. BiP does not pull the protein through; it just stops it from sliding back. (The reason why BiP is required for post-translational translocation but not for co-translational translocation may be that a newly synthesized protein is continuously extruded from the ribosome and therefore cannot slip backward.)