S

subunit

▲ EXPERIMENTAL FIGURE 16-8 Electron microscopy reconstruction reveals that a translocon associates closely with a ribosome. Purified Sec61 complexes were solubilized by treatment of ER membranes with detergents. When ribosomes were added, translocons (blue) reassembled in artificial phospholipid bilayers. The resulting particles were frozen, and electron micrographs of a large number of particles were generated, stored in a computer, and then averaged to produce a single image. A representation of the approximate size and position of the ER lipid bilayer has been added. Note that although the ribosome is firmly attached to the translocon, there is a gap between the two structures. The fingerlike appendage below the translocon channel is thought to be formed from a protein complex that associates with the translocon. [Courtesy Dr. Christopher Akey and Jean-Francois Menetret, Boston University School of Medicine.]

sequence and approximately 30 adjacent amino acids, can insert into the translocon pore (see Figure 16-6).

The mechanism by which the translocon channel opens and closes is controversial at this time. Some evidence suggests that a protein within the ER lumen blocks the translo-con pore when a ribosome is not bound to the cytosolic side of the translocon. Other observations, however, indicate that Sec61 complexes may normally reside in the ER membrane in an unassembled state and that the gating process involves the assembly of a translocon channel at the site where the ribosome and nascent chain are brought to the membrane by the SRP and SRP receptor.

As the growing polypeptide chain enters the lumen of the ER, the signal sequence is cleaved by signal peptidase, which is a transmembrane ER protein associated with the translo-con (see Figure 16-6). This protease recognizes a sequence on the C-terminal side of the hydrophobic core of the signal peptide and cleaves the chain specifically at this sequence once it has emerged into the luminal space of the ER. After the signal sequence has been cleaved, the growing polypep-tide moves through the translocon into the ER lumen. The translocon remains open until translation is completed and the entire polypeptide chain has moved into the ER lumen.

ATP Hydrolysis Powers Post-translational Translocation of Some Secretory Proteins in Yeast

In most eukaryotes, secretory proteins enter the ER by co-translational translocation, using energy derived from translation to pass through the membrane, as we've just described. In yeast, however, some secretory proteins enter the ER lumen after translation has been completed. In such post-translational translocation, the translocating protein passes through the same Sec61 translocon that is used in co-translational translocation. However, the SRP and SRP receptor are not involved in post-translational translocation, and in such cases a direct interaction between the translo-con and the signal sequence of the completed protein appears to be sufficient for targeting to the ER membrane. In addition, the driving force for unidirectional translocation across the ER membrane is provided by an additional protein complex known as the Sec63 complex and a member of the Hsc70 family of molecular chaperones known as BiP. The tetrameric Sec63 complex is embedded in the ER membrane in the vicinity of the translocon, while BiP is localized to the ER lumen. Like other members of the Hsc70 family, BiP has a peptide-binding domain and an ATPase domain. These chaperones bind and stabilize unfolded or partially folded proteins (see Figure 3-11).

The current model for post-translational translocation of a protein into the ER is outlined in Figure 16-9. Once the N-terminal segment of the protein enters the ER lumen, signal peptidase cleaves the signal sequence just as in cotrans-lational translocation (step 1). Interaction of BiP-ATP with the luminal portion of the Sec63 complex causes hydrolysis of the bound ATP, producing a conformational change in BiP that promotes its binding to an exposed polypeptide chain (step 2). Since the Sec63 complex is located near the translo-con, BiP is thus activated at sites where nascent polypeptides can enter the ER. Certain experiments suggest that in the absence of binding to BiP, an unfolded polypeptide slides back and forth within the translocon channel. Such random sliding motions rarely result in the entire polypeptide's crossing the ER membrane. Binding of a molecule of BiP-ADP to the luminal portion of the polypeptide prevents backsliding of the polypeptide out of the ER. As further inward random sliding exposes more of the polypeptide on the luminal side of the ER membrane, successive binding of

Translocating polypeptide chain

ER lumen

Translocating polypeptide chain

ER lumen

Cleaved signal sequence

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