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The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Since the identification of essential protein-translocation components in the endoplasmic reticulum membrane, research efforts have concentrated on the elucidation of the molecular mechanism of protein transport across this membrane. Recent results have provided new information as to how proteins are targeted to, and inserted into, the translocation site during translation. Post-translational translocation has also been examined and is distinct from cotranslational translocation with respect to the mechanism and membrane protein components involved.

The signal-recognition particle (SRP) is important for the targeting of many secretory and membrane proteins to the endoplasmic reticulum (ER). Targeting is regulated by three GTPases, the 54K subunit of SRP (SRP54), and the alpha- and beta-subunits of the SRP receptor. When a signal sequence emerges from the ribosome, SRP interacts with it and targets the resulting complex to the ER membrane by binding to the SRP receptor. Subsequently, SRP releases the signal sequence into the translocation channel. Here we use a complex of a ribosome with a nascent peptide chain, the SRP and its receptor, to investigate GTP binding to SRP54, and GTP hydrolysis. Our findings indicate that a ribosomal component promotes GTP binding to the SRP54 subunit of SRP. GTP-bound SRP54 is essential for high-affinity interaction between SRP and its receptor in the ER membrane. This interaction induces the release of the signal sequence from SRP, the insertion of the nascent polypeptide chain into the translocation channel, and GTP hydrolysis. The contribution of the ribosome had previously escaped detection because only synthetic signal peptides were used in the analysis.

Ricin A chain is a polypeptide of 267 amino acids containing a hydrophobic region near its carboxyl-terminus (residues 245-256) which has been implicated in the membrane translocation step necessary for this catalytically active toxin to reach its intracellular substrate. DNA fusions were constructed that encoded hybrid proteins consisting of carboxyl-terminal residues 233-267 or residues 238-267 of ricin A chain preceding mouse dihydrofolate reductase. When in vitro transcripts prepared from these constructs were translated in cell-free systems, the ricin A chain-derived sequences functioned as efficient signal peptides which directed dihydrofolate reductase into microsomes or into proteoliposomes containing microsomal membrane components.

We have analyzed early phases of the cotranslational transport of the secretory protein preprolactin through the mammalian endoplasmic reticulum (ER) membrane. Following recognition of the signal sequence of the nascent polypeptide chain in the cytosol by the SRP, the chain is transferred into the membrane, where a second signal sequence recognition step takes place for which the presence in the lipid bilayer of the Sec61p complex is essential and sufficient. This step leads to a tight junction between the ribosomenascent chain complex and the Sec61p complex, and to the productive insertion of the nascent chain into the translocation site. These results show that a translocation substrate is subjected to two recognition events before being allowed to cross the ER membrane.

Protein transport across the endoplasmic reticulum membrane in eukaryotes and across the cytoplasmic membrane in bacteria have turned out to be highly related. The core component of the translocation apparatus is the Sec61/SecYp complex; at least two of its subunits are conserved in evolution. The Sec61/SecYp complex is involved in both co- and post-translational transport pathways. The two modes require probably distinct additional components.