The Signal Recognition Particle Receptor Is a Complex That Contains Two Distinct Polypeptide Chains

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69kD polypeptide (Qt-subunit, SRet), a 30-kD 13-subunit (SRJ]). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are ~30 kD in size and are immunologically distinct from each other, as well as from SRa and SRP proteins. One of the 30-kD proteins (SR~) forms a tight complex with SR~t in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SRct or SR~. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SRI3 is an integral component of SRP receptor. The presence of SRI~ was previously masked by proteolytic breakdown products of SRct observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SRct. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SRa and SRI3. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation. N " ASCENT secretory proteins are targeted specifically to the endoplasmic reticulum (ER) ~ membrane. Two components, the signal recognition particle (SRP) and the SRP receptor (or docking protein [23]), are known constituents of the cellular targeting apparatus responsible for this protein sorting event (30). SRP binds to signal sequences within the nascent polypeptide chain as it emerges from the ribosome (18, 19) and causes an arrest or pause of protein synthesis. Then, when the ribosome-bound SRP interacts with the ER membrane, the elongation arrest is released (27). The ribosome engages in a functional ribosome-membrane junction that translocates the growing polypeptide chain across the membrane by a mechanism that is, as yet, poorly understood. The SRP receptor was functionally defined as an activity residing in a microsomal membrane protein that would release the elongation arrest (27). This activity was purified by SRP-Sepharose affinity chromatography and attributed to a 69-kD ER membrane protein (12), which we will henceforth refer to as the Q-subunit of the SRP receptor (SRet). Independent evidence for the involvement of SRt~ in protein translocation was provided by proteolytic dissection experiments. Treatment of microsomal membranes with a variety of proteases leads to the release of a 52-kD cytoplasmic domain of ~1. Abbreviations used in this paper: ER, endoplasmic reticulum; mp30, 30kD ER membrane protein; SRtt and SRI3, ¢tand 13-subunits of the SRP receptor; SRP, signal recognition particle. SRa from the membrane and a concomitant loss of the ability of these membranes to translocate secretory proteins. Readdition of the purified 52-kD cytoplasmic domain to proteolyzed microsomes will reconstitute functional SRP receptor and restore the translocation activity of the vesicles (22, 31). Recently, we determined the primary sequence of SRct from cDNA clones. We established that SRct is anchored to the ER membrane by its amino-terminal region and that the membrane anchor fragment and the 52-kD cytoplasmic domain jointly contribute to a functionally important region, which is highly charged and may function as the SRP binding site (20). Here we report that the SRP receptor contains an additional subunit of •30 kD that has not been separated from SRtt in previous studies. Materials and Methods