ATTACHMENT OF RIBOSOMES TO MICROSOMAL MEMBRANES

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The long-term objective of this project is to understand the biogenetic mechanisms responsible for the subcellular distribution of specific proteins within membranes and organelles. The biosynthesis of several model proteins with diverse destinations, including the endoplasmic reticulum (e.g., cytochrome P-450 and its reductase), plasma membrane (e.g., erythrocyte band 3, the sodium-potassium ATPase and viral envelope glycoproteins), lysosomes (e.g., cathepsin D and b-glucuronidase) and mitochondria (e.g., cytochrome C and cytochrome oxidase) will be studied. For proteins which are synthesized in bound polysomes, features which serve as signals for their cotranslational insertion into the ER or which halt the vectorial discharge and lead to retention of polypeptides within the membranes will be identified. This will be accomplished using in vitro protein synthesis systems with added microsomal membranes and by analysis of the primary amino acid sequences derived from protein chemistry and cDNA sequencing studies. It is expected that the number and location of insertion and halt transfer signals in a membrane polypeptide will account for its transmembrane disposition. Sorting out processes which, following cotranslational insertion of a polypeptide into the ER, ensure its distribution within the cell will be investigated. Cytochemical studies on Golgi membranes will be carried out to relate the polarity of the Golgi complex to its role in sorting out mechanisms. The pathway of a polypeptide will be stutied using agents which perturb the transfer of products between organelles within cells and in vitro reconstruction systems for this transfer will be developed. The assignment of a specific role to polypeptide segments in the insertion, halt transfer or sorting processes will be aided by recombinant DNA procedures to construct modified and chimeric genes which will be introduced into cultural cells to follow the distribution of the products of their expression. Post-translational incorporation of polypeptides into mitochondria will also be investigated to study the mechanism of action of an addressing signal which was identified within cytochrome C and directs the uptake of this polypeptide into the organelle.