Interaction of nascent polypeptides with signal recognition particle

Abstract

Most secretory proteins are synthesized as precursors possessing a transient NH{dollar}\sb2{dollar}-terminal signal peptide, which mediates precursor binding to and translocation across the endoplasmic reticulum (ER) membrane. The signal recognition particle (SRP) binds to the ribosome-nascent precursor complex and inhibits further translational elongation. The complex is targeted to the ER membrane by binding to the SRP receptor. Translational arrest is then released and protein synthesis is completed concomitant with translocation across the ER membrane. Currently, the precise mechanism of signal peptide - SRP interaction and of translational arrest are poorly understood. The goals of this project therefore were: (i) to investigate signal peptide - SRP interactions by determining the size constraints for a polypeptide to be susceptible to SRP-mediated inhibition; and (ii) to determine the mechanism of insertion of small polypeptides into the ER membrane. Preproinsulin was employed as a model for these experiments.;Recombinant DNA molecules encoding a series of COOH-terminally truncated preproinsulin molecules that possess the same NH{dollar}\sb2{dollar}-terminal domain as preproinsulin were transcribed and the mRNA translated in vitro in the presence or absence of SRP. The precursors were 116 amino acids (preproinsulin), 78 amino acids (78-mer), 64 amino acids (64-mer) and 45 amino acids (45-mer). Preproinsulin, 78-mer, and 64-mer were arrested by SRP, but the 45-mer was not susceptible to SRP arrest. The size of the smallest nascent polypeptide that could be arrested by SRP was estimated to be 50 amino acids.;The degree of inhibition of protein synthesis by SRP was proportional to precursor size; preproinsulin was most inhibited by SRP whereas the 64-mer was least inhibited. Furthermore, SRP induced a time lag in completion of translation that was directly proportional to precursor size.;The mechanism of membrane insertion of the truncated preproinsulin molecules was also studied to investigate the possibility that small polypeptides insert into the ER by an SRP-independent, post-translational mechanism. Insertion of the preproinsulin truncations was demonstrated to be absolutely dependent on the presence of SRP and on the polypeptides being tethered to ribosomes during the insertion process. (Abstract shortened with permission of author.).