Many proteins which are synthesized in the cytoplasm of mammalian cells must then cross various intracellular organelle membranes and be sequestered in the compartments where their physiologic functions are performed. This research proposal is aimed at elucidation of the molecular events involved in the translocation of these newly synthesized proteins across membranes and definition of the co-translational and post-translational modifications which occur during this process. Specifically, we will utilize in vitro translation of mRNAs encoding the hormone, human placental lactogen; the secretory protein, rat liver prothrombin; and the mitochondrial enzymes, glutamate dehydrogenase and malate dehydrogenase, to study the translocation of these cytoplasmically-synthesized proteins across endoplasmic reticular and mitochondrial membranes. We will purify and characterize some of the membrane components involved in translocation including the signal peptidase which removes the pre-peptide from secretory proteins during their translocation. These studies will define some of the events involved in compartmentalization of proteins and will provide basic information necessary to understand regulation of the membrane translocation process.