Eucaryotic cells contain within their plasma membranes many unique compartments and organelles. The localization of specific proteins to each organelle is essential for the function of the subcellular compartment and for the maintenance of normal cellular physiology; despite their importance, the biochemical mechanisms by which proteins are targeted and delivered to their destinations are largely unknown. To define the cellular machinery involved in the trafficking of proteins, the transport of acid hydrolases to lysosomes is being studied. Acid hydrolases receive a recognition marker, mannose 6-phosphate (Man 6-P), after their synthesis which directs the enzymes to lysosomes. To learn where the phosphorylation reaction occurs, the biogenesis of a single acid hydrolase, Beta-glucuronidase, will be studied in mouse lymphoma cells. The cells will be metabolically labeled with 3H-mannose and the post-translational processing of the enzyme's oligosaccharides will be analyzed following immunoprecipitation of the radiolabeled molecules from cell extracts. The phosphorylated oligosaccharides mediate binding of the acid hydrolases to either of two Man 6-P binding proteins; a 215 kD cation-independent and a 46 kD cation-dependent form. An investigation will be conducted to determine: 1) if both Man 6-P binding proteins mediate the delivery of Man 6-P-containing ligands to lysosomes, and 2) if the two """"""""receptors"""""""" transport different ligands. To delineate the intracellular pathway taken by the receptor-ligand complexes en route to lysosomes, the location of the enzymes responsible for the removal of the Man 6-P recognition marker will be analyzed. An in vitro assay will be developed to identify and characterize the degradative enzyme(s); specific phosphorylated oligosaccharides containing one or two phosphomonoesters will be evaluated as substrates. The assay will be used to localize the mannose 6-phosphatase within isolated subcellular organelles. The location of the processing phosphatase will also be investigated by identifying the subcellular compartment containing acid hydrolases internalized by cells at 20 Degree C; under these conditions, molecules entering cells via receptor-mediated endocytosis do not reach lysosomes yet they encounter a phosphatase. The compartment containing the internalized enzymes will be examined with respect to its morphology and its biochemical capabilities. It is hoped that these studies will lead to a better understanding of protein trafficking and provide insight into the mechanisms by which other recognition events occur during development and differentiation.
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