Translocation across or integration into the rough endoplasmic reticulum (RER) membrane is the first step in the intracelluar sorting of proteins in eukaryotic cells. This process is initiated when a signal sequence in the nascent protein chain emerges from the ribosome and is recognized by the signal recognition particle (SRP). The resulting SRP-ribosome-nascent chain-complex (SRP-RNC) is targeted to the RER membrane through the concerted action of the SRP and the SRP receptor (SR). The nascent chain is then displaced from SRP and transferred to the translocon, a proteinaceous channel composed of oligomers of the Sec61 complex. To gain a better understanding of the molecular mechanism of protein translocation, we treated ribosome-stripped micro somes with proteases of different cleavage specificities to sever cytoplasmic domains of SRα, SRβ, TRAM, and the Sec61 complex, and then characterized protein translocation intermediates that accumulate when Sec61α or SRβ is inactivated by proteolysis. We found that GTP hydrolysis by the SRα-SRP complex and dissociation of SRP54 from the signal sequence are blocked in the absence of a functional Sec61 complex. Experiments using SR-reconstituted proteoliposomes confirmed the assembly of a membrane-bound, GTP-stabilized post-targeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54. This regulatory role of Sec61α is proposed to provide a mechanism that inhibits signal sequence dissociation from SRP54 if the adjacent Sec61 complex is occupied by a translating ribosome, thereby insuring efficient transfer of an RNC from the SRP-SR complex to the translocation channel.

We also found that complex formation between SRα and SRP is compromised in the absence of intact SRβ. Results obtained using a soluble system of in vitro translated SRα and SRβ suggest that SRβ is either required for GTP binding to SRα and SRP54 or for stabilizing the SRα-SRP complex. Moreover, using the XTP mutants of SRα and SRP54, we found that XTP cannot support efficient protein translocation in the absence of GTP. The addition of GTP dramatically promotes protein translocation into the endoplasmic reticulum, suggesting the GTPase activity of SRβ is required for this process. Further mutagenesis experiments revealed that the GTP-binding pocket of SRβ is involved in dimerization with SRa. All these data demonstrate that SRβ is important in protein translocation and will help elucidate the precise role of SRβ in vivo.