Signal Recognition Protein Is Required for the Integration of Acetylcholine Receptor 8 Subunit, a Transmembrane Glycoprotein, into the Endoplasmic Reticulum Membrane

Purified Signal Recognition Protein (SRP) has previously been shown to be required for the translocation of secretory proteins across the microsomal membrane (Walter and Blobel, 1980 . Proc. Nat/ . Acad. Sci. U . S. A. 77:7,112-7,116) and to function in the early events of this process (Walter and Blobel, 1981. J. Cell Biol . 91 :557-561) . We demonstrate here that the S subunit of acetylcholine receptor (AChR-S), a transmembrane glycoprotein, likewise requires SRP for its asymmetric integration into microsomal membranes. We further demonstrate by partial sequence analysis that AChR-8 is synthesized with a transient NH2-terminal signal sequence of 21 residues that is cleaved off during integration into microsomal membranes . Integration of AChR-S into the microsomal membrane vesicles proceeded asymmetrically, yielding a large (44 kdalton) core-glycosylated domain, inaccessible to externally added proteolytic enzymes and a smaller (-16 kdalton) domain exposed on the outside of the vesicles and accessible to externally added proteolytic enzymes. The NH2 terminus of the molecule is contained in the 44-kdalton domain . For numerous integral membrane proteins, it appears that the endoplasmic reticulum (ER) is the exclusive site of asymmetric membrane integration and hence the exclusive port of entry into the membranes of all ER-derived organelles . The ER's capacity for asymmetric integration of proteins has long been assumed to be related to its ability to translocate secretory proteins (1, 2) . Experimental evidence in support of this conjecture was the observed competition (3) between nascent chains of a secretory protein (bovine preprolactin) and of a viral transmembrane protein (glycoprotein G of vesicular stomatitis virus) for a common translocation "site" in the microsomal membranes (dog pancreas) that were present in a cellfree translation system . Recently, we have accomplished a partial characterization of the ER's translocation activity . From a 0.5 M salt extract of microsomal membranes we have purified a protein (4) termed Signal Recognition Protein (SRP) that is required to restore translocation competence to the salt-extracted microsomal membrane (5, 6) . SRP appears to function in mediating the engagement of polysomes synthesizing secretory proteins with the microsomal membrane (6). Most interestingly, in the absence of microsomal membranes SRP was shown to selectively inhibit translation of mRNA's for secretory proteins (5) by a signal sequence-induced, site-specific arrest ofchain elongation (7) . This elongation arrest was reversible and was released when salt-extracted microsomal vesicles were added to the cellfree translation system (7) . THE JOURNAL OF CELL BIOLOGY " VOLUME 93 MAY 1982 501-506 © The Rockefeller University Press " 0021-9525/82/05/0501/06 $1 .00 To investigate whether in fact translocation of secretory proteins and integration of transmembrane proteins share identical initial steps we used the recently accomplished (8) in vitro integration into dog pancreas microsomal membranes ofthe S subunit of the acetylcholine receptor (AChR-S) of Torpedo californica as a model system . As in the case of secretory proteins (5, 9) we found that AChR-S synthesis in a cell-free wheat germ system was inhibited by SRP, that addition of microsomal membranes (saltextracted) released this inhibition, and that SRP was required to accomplish integration of AChR-S into such microsomal membranes. We have also demonstrated that AChR-S is synthesized with an NH2-terminal signal sequence that was correctly cleaved upon asymmetric integration of AChR-8 into the heterologous microsomal membranes. The NH2 terminus ofthe integrated form of AChR-S was resistant to cleavage by externally added proteases, although this proteolysis removed almost 16 kdaltons from the molecule . MATERIALS AND METHODS