The proper insertion of the nascent polypeptide chains with the short n (polar) and/or h (hydrophobic) regions in their signal sequences requires involvement of the TRAM protein, which is as abundant in the endoplasmic reticulum membrane as the Sec61p complex. The TRAM protein is a 34 kDa glycoprotein presumably spanning the membrane eight times (as predicted from its amino acid sequence) with hydrophilic and charged residues located in the membrane interior, similarly to Sec61a.

The morphology of the translocation channel was studied by both the traditional electron microscopy with negative staining (Hanein etal., 1996) and the cryo-electron microscopy (Beckmann et al., 1997). It has been found that the Sec61p heterotrimers in one projection appear as rings with characteristic pentagonal contours of about 85 A in diameter and with a central pore of about 20 A. The rings (or, more exactly, pentagons) are shown to represent a top view of cylinders (or, more correctly, toroidal structures) of 50 to 60 A in height. By applying the cylinder model and taking the dimensions into account, the "molecular mass" of the particle was estimated to be of about 250 kDa. Hence, several (probably, five) Sec61p heterotrimers appear to participate in the formation of the particle.

Interestingly, the rings are not seen in the proteoliposomes containing the Sec61p heterotrimers. However, they arise after addition of ribosomes that play a role of ligands stimulating specific association of the heterotrimers. Similar pentagonal rings can be identified on the surface of microsomes (freeze-

fracture electron microscopy). There are good reasons to believe that Sec61p is an obligatory constituent of such structures. The microsomal rings are larger than those in the detergent-solubilized Sec61p preparations (100 A versus 85 A in diameter), that may be either owing to different conformation of the Sec61p oligomers or because of the involvement of some additional subunits (e.g., the TRAM protein).

Thus, the pentagon-shaped toroidal particles formed by the Sec61p heterotrimers are likely to be the protein-translocating channels in the endoplasmic reticulum membrane. The involvement of several heterotrimers in the functioning of such channel, along with potentially amphiphilic properties of Sec61p(a), allows the coexistence of hydrophobic and hydrophilic sections of a growing polypeptide chain in the channel (see below).

Figure 18.9 represents a model for assembly of the translocation channel composed of the Sec61p heterotrimers. According to this model, in the endoplasmic reticulum membrane, monomeric and oligomeric forms of the heterotrimer are in an equilibrium shifted towards the former ones. The ribosomes favor the formation of the oligomeric cylindrical particles that are still closed at the end (assembly stage). While the ribosome with a nascent polypeptide chain is sitting on the cytoplasmic end of the particle, its interior opens itself thus allowing the polypeptide chain to penetrate through the membrane (gating stage).


disassembly equilibrium




Figure 18.9. Tentative sequence of events during formation of the translocating channel through the membrane.

(1) In the absence of ligands there is an equilibrium between monomeric and oligomeric forms of the membrane protein Sec61p (assembly-disassembly equilibrium), shifted towards the monomeric form.

(2) The binding of the ribosome/nascent polypeptide complex (see Fig. 18.8) stimulates the assembly of the Sec61p oligomer in the membrane.

(3) The interaction of the Sec61p oligomer with the signal sequence of the nascent polypeptide chain opens the channel for transmembrane translocation (gating stage).

(D. Hanein, K. E. S. Matlack, B. Jungnickel, K. Plath, K.-U. Kalies, K. R. Miller, T. A. Rapoport & C. W. Akey, Cell 87, 721-732, 1996).

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