In vitro assembled yeast ribosome-nascent chain complexes (RNCs) containing a signal sequence in the nascent chain were immunopurified and reconstituted with the purified protein-conducting channel (PCC) of yeast endoplasmic reticulum, the Sec61 complex. A cryo-EM reconstruction of the RNC-Sec61 complex at 15.4 A resolution shows a tRNA in the P site. Distinct rRNA elements and proteins of the large ribosomal subunit form four connections with the PCC across a gap of about 10-20 A. Binding of the PCC influences the position of the highly dynamic rRNA expansion segment 27. The RNC-bound Sec61 complex has a compact appearance and was estimated to be a trimer. We propose a binary model of cotranslational translocation entailing only two basic functional states of the translating ribosome-channel complex.

Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the ␣-and ␤-subunits of the SRP receptor (SR). SR␤ is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SR␤-GTP in complex with the binding domain of SR␣ suggested that SR␤, like all ARF-type GTPases, requires a guanine nucleotide exchange factor (GEF) for function. Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the ␤-subunits of the two homologous heterotrimeric protein-conducting channels in yeast. Using a fluorescence nucleotide exchange assay, we show that the ␤-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SR␤. Both the cytosolic domain of Sec61␤ as well as the holo-Sec61␤, when part of the isolated trimeric Sec61p complex, function as the GEF for SR␤, whereas the same Sec61␤, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SR␤.Of the GTPases involved in cotranslational protein transport, the signal recognition particle (SRP) 1 subunit SRP54 recognizes and binds to the signal sequence of the nascent chain, presented by the ribosome nascent chain complex (RNC) (1). The interaction between SRP54 and the membrane bound SRP receptor (SR) selectively targets the RNC-SRP complex to the membrane of the endoplasmic reticulum (ER). This is followed by the release of the signal sequence from SRP54 and its insertion into the protein-conducting channel (PCC) that is formed by the oligomeric assembly of the trimeric Sec61p complex (2). The nascent chain translocates into the ER lumen, while the ribosome remains attached to the PCC.The GTPase domains of SRP54 and SR␣ are very similar in their structure and function. Structural studies of their prokaryotic homologues have shown that they form their own family within the GTPase superfamily (3, 4). They have low affinity for nucleotide and are both stable in their empty states (5, 6). SRP54 binds GTP when in contact with the RNC (6). The interaction between SRP54 and SR␣ leads to the formation of a GTP stabilized SRP-SR␣ complex (7). GTP hydrolysis dissociates the SRP-SR␣ complex (8), whereby SRP54 and SR␣ serve as mutual GTPase-activating proteins (GAP). This reciprocally symmetric interaction is unique among known GTPases (9).SR␤ is closely related to members of the ADP-ribosylation factor (ARF) GTPase family (10, 11). ARFs are conserved in all eukaryotes and are involved in the regulation of vesicle transport (12). SR␤ is present only in eukaryotes and contains a transmembrane segment providing the membrane anchor for SR␣ (13). However, a truncated protein representing the cytosolic GTPase domain without the transmembrane domain is functional in protein targeting (14). The function of the GTPase domain of SR␤ has long been a matter ...

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