Translocation of Proteins Across the Endoplasmic Reticulum

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin) . This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the saltextract of microsomal vesicles (Walter and Blobel, 1980 . Proc. Nat/ . Acad . Sci. U. S. A. 77:7112-7116) . SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles . Likewise, polysome binding to the microsomal membrane was largely abolished when 8-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides . The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions . In the preceding paper (1) we have described the effects of signal recognition protein (SRP) in a wheat germ cell-free translation system programmed with mRNA's for either rabbit globin (cytoplasmic protein) or bovine prolactin (secretory protein) . We have demonstrated (1) that SRP binds specifically to monomeric ribosomes, albeit with relatively low affinity (apparent kD < 5 x 10-5 M), and that it binds selectively and with 6,000-fold higher affinity (apparent kD < 8 x 10-9 M) to in vitro assembled polysomes synthesizing secretory protein (but not to those synthesizing globin). This 6,000-fold enhancement most likely results from specific recognition by SRP of the signal sequence of the nascent secretory polypeptide . In this paper we describe the specific effects of SRP in a wheat germ cell-free translation system that was supplemented with dog pancreas microsomal membrane vesicles . Our data show that SRP mediates the selective binding to microsomal membranes of nascent, in vitro assembled polysomes synthesizing secretory protein but not of those synthesizing cytoplasmic protein . Polysome binding is abolished if SRP is modified THE JOURNAL OF CELL BIOLOGY VOLUME 91 NOVEMBER 1981 551-556 ©The Rockefeller University Press 0021-9525/81/11/0551/06 $1 .00 by N-ethyl maleimide (NEM) or if the nascent secretory polypeptide is modified by incorporation ofa-hydroxy leucine, an analogue of Leu. MATERIALS AND METHODS The preparation of various microsomel membrane fractions (RM, K-RM), the extraction and purification of SRP, the cell-free wheat germ translation system, and the quantitation of in vitro synthesized protein were described in the preceding paper(1). The SRP preparation used was the eluate oftheaminopentylagarose resin, except when stated otherwise . Assay for Binding of In Vitro Assembled Polysomes to Microsomal Membranes The assay for nascent polysome binding to microsomal membranes was an indirect one. We measured the depletion ofmRNA from the translation system resulting from recruitment into membrane-bound polysomes and removal of these in vitro assembled rough microsomes (RM) by differential centrifugation. The initial incubation volume for each timepoint was 75 dal. The wheat germ cell-free translation system plus additional components (specified in figure leg551 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published November 1, 1981