October 18, 2012

The translocon forms a pore

  • A translocon is a discrete structure in a membrane that forms a channel through which (hydrophilic) proteins may pass.
  • The Sec61 trimeric complex provides the channel for proteins to pass through a membrane.
  • A translocating protein passes directly from the ribosome to the translocon without exposure to the cytosol. 

There is a basic problem in passing a (largely) hydrophilic protein through a hydrophobic membrane. The energetics of the interaction between the charged protein and the hydrophobic lipids are highly unfavorable. However, a protein in the process of translocation across the ER membrane can be extracted by denaturants that are effective in an aqueous environment. The same denaturants do not extract proteins that are resident components of the membrane. This suggests the model for translocation illustrated in Figure 8.26, in which proteins that are part of the ER membrane form an aqueous channel through the bilayer. A translocating protein moves through this channel, interacting with the resident proteins rather than with the lipid bilayer. The channel is sealed on the lumenal side to stop free transfer of ions between the ER and the cytosol.
The channel through the membrane is called the translocon. Its components have been identified in two ways. Resident ER membrane proteins that are crosslinked to translocating proteins are potential subunits of the channel (Mothes, Prehn, and Rapoport, 1994). And sec mutants in yeast (named because they fail to secrete proteins) include a class that cause precursors of secreted or membrane proteins to accumulate in the cytosol (Deshaies and Schekman, 1987; Esnault et al., 1993). These approaches together identified the Sec61 complex, which consists of three transmembrane proteins: Sec61α,β,γ. Sec61 is the major component of the translocon. In detergent (which provides a hydrophobic milieu that mimics the effect of a surrounding membrane), Sec61 forms cylindrical oligomers with a diameter of ~85Å and a central pore of ~20Å. Each oligomer consists of 4 heterotrimers (Hanein et al., 1996).
Is the channel a preexisting structure (as implied in the figure) or might it be assembled in response to the association of a hydrophobic signal sequence with the lipid bilayer? Channels can be detected by their ability to allow the passage of ions (measured as a localized change in electrical conductance). Ion-conducting channels can be detected in the ER membrane, and their state depends on protein translocation (Simon and Blobel, 1991; Crowley, 1994). This demonstrates that the channel is a permanent feature of the membrane.
A channel opens when a nascent polypeptide is transferred from a ribosome to the ER membrane. The translocating protein fills the channel completely, so ions cannot pass through during translocation. But if the protein is released by treatment with puromycin, then the channel becomes freely permeable. If the ribosomes are removed from the membrane, the channel closes, suggesting that the open state requires the presence of the ribosome. This suggests that the channel is controlled in response to the presence of a translocating protein.
Measurements of the abilities of fluorescence quenching agents of different sizes to enter the channel suggest that it is large, with an internal diameter of 40-60Å. This is much larger than the diameter of an extended α-helical stretch of protein. It is also larger than the pore seen in direct views of the channel; this discrepancy remains to be explained (Simon and Blobel, 1991).
The aqueous environment of an amino acid in a protein can be measured by incorporating variant amino acids that have photoreactive residues. The fluorescence of these residues indicates whether they are in an aqueous or hydrophobic environment. Experiments with such probes show that when the signal sequence is first synthesized in the ribosome, it is in an aqueous state, but is not accessible to ions in the cytosol. It remains in the aqueous state throughout its interaction with a membrane. This suggests that the translocating protein travels directly from an enclosed tunnel in the ribosome into an aqueous channel in the membrane.

In fact, access to the pore is controlled (or "gated") on both sides of the membrane. Before attachment of the ribosome, the pore is closed on the lumenal side. Figure 8.27 shows that when the ribosome attaches, it seals the pore on the cytosolic side. When the nascent protein reaches a length of ~70 amino acids, that is, probably when it extends fully across the channel, the pore opens on the lumenal side. So at all times, the pore is closed on one side or the other, maintaining the ionic integrities of the separate compartments (Crowley, 1994; Liao et al., 1997).
The translocon is versatile, and can be used by translocating proteins in several ways: