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18.5.2.Insertion of the Nascent Polypeptide Chain into the Endoplasmic Reticulum Membrane

As indicated above, after dissociation from SRP, the ribosome/nascent chain complex remains attached to the endoplasmic reticulum membrane, the nascent chain being inserted into the translocation channel. Such insertion, however, is not tightly coupled to the SRP/SR interaction. In fact, proteoliposomes containing Sec61p as the only protein constituent bind the ribosome/short nascent chain (80-90 residues) complexes in a signal-sequence-dependent manner, whereupon the polypeptide chains become protease-resistant. Mutations in the hydrophobic h region of the signal sequence result in disturbance of such binding. Thus, the signal sequence is recognized at least twice: first, when polypeptide chains to be exocyted are selected by means of their binding with SRPs; second, when they enter into the translocation channel. In other words, a cell exercises double control over penetration of polypeptide chains into the endoplasmic reticulum and further into the exocytic pathway. Thus, although the nascent polypeptide chain can interact directly with the translocation channel, as it follows from the model experiments with proteoliposomes, the interaction is most commonly mediated by SRP and SR in the case of natural microsomes.

As to the ribosomal moiety of the ribosome/ nascent chain complex, it binds tightly to the translocation channel, most probably by direct interaction with the Sec61p complex. It has been found that the Sec61a protein can be detached from ribosomes, preliminary freed of nascent polypeptide chains by treatment with puromycin, only in the presence of detergents (e.g., digitonin) and concentrated (ca. 1.2 M) salt solutions. Thus, Sec61a falls into the category of the endoplasmic reticulum membrane proteins most firmly bound to the ribosomes. Currently, the involvement of other membrane proteins in the ribosome binding (e.g., putative 34 kDa and 180 kDa ribosome receptors) is debatable.

It is believed that the nascent chain penetrates into the channel in the form of a loop exposing the polar (usually positively charged) amino terminus to the cytoplasm, with both the signal sequence and the mature part contacting the Sec61p protein until cleavage of the signal sequence by the signal peptidase (Fig. 18.10 A). The nascent chains with negatively charged amino-terminal residues probably do not form the loop structure in the translocation channel. Their amino termini face the lumen of the endoplasmic reticulum while the signal sequences span the membrane once, thereupon playing the role of an anchor (Fig. 18.10 B). Such an arrangement of the nascent chain in the channel can be changed to the loop-shaped one just by substituting positively charged amino-terminal residues for negatively charged ones.

As a result of the interaction of the nascent polypeptide chain with the endoplasmic reticulum membrane, the ribosome renders attached to the channel structure in a manner that it seals up the

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