This is a proposal to identify and characterize membrane components of the rough endoplasmic reticulum and cytoplasmic factors which are involved in the recognition and binding of ribosomes and signal sequences in nascent polypeptide chains and may function as a vectorial discharge apparatus. Undenatured ribophorins and cytoplasmic protein of 93,000 daltons, will be purified by an affinity chromatography procedure based on the specific interaction of these proteins with the putative signal segments which are present in ovalbumin and in some mature membrane proteins, such as cytochrome P450 and epoxide hydratase. The affinity purified native proteins will be incorporated into the membranes of reconstituted vesicles which will be used in vitro assays to identify their functions in specific steps of the vectorial discharge. The effect of specific antiboides (monoclonal) against purified microsomal proteins on discrete steps of the cotranslational discharge of nascent polypeptides will be assessed in in vitro assays. Information on the structure and spatial dispositions of the ribophorins will be obtained using protein chemistry procedures, immunocytochemistry and sequencing of DNA's complementary to ribophorin mRNA's. Radioimmunoassays will be developed to establish correlations between the ribophorin content of microsomal membranes and their translocation activity during the development of the endoplasmic reticulum. The biosynthesis of ribophorins and the mechanisms for their insertion and retention in endoplasmic reticulum membranes will be studied using in vitro and in vivo systems. A possible role of the Golgi apparatus in the post-translational processing of these glycoproteins will be assessed.
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