): Biogenesis of a membrane protein is a dynamic process with multiple steps occurring in parallel. Defects in membrane protein biosynthesis are manifested in several genetic diseases, and may be pivotal to understanding many more. However, before lesions of membrane protein biogenesis can be discovered, mechanisms which describe the fate of a membrane protein must first be found. Using the multipass protein opsin, experiments are proposed which separate translation, membrane targeting, translocation, and integration. The ability to separate the various steps of opsin biogenesis hinges on the observation that mRNAs that lack stop codons are translated by the ribosome but are not released. In the case of translation of truncated mRNAs for opsin, the arrested ribosomes with their nascent polypeptides bound are still capable of interacting with the translocation machinery in vitro. These truncated proteins can undergo targeting, translocation, and integration as assayed by signal recognition peptide (SRP) binding and glycosylation. Using the unique biochemical properties of arrested ribosomes three experimental aims will be addressed. First, when does the signal recognition particle interact with the first transmembrane domain of opsin? Second, what is the minimum amount of sequence required to support targeting and translocation of the nascent chain? Third, when during translation is opsin competent to translocate across the ER? It is hoped that answers to these questions will be more generally applicable to the biosynthesis of other multiple pass proteins.
