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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033342-04
Application #
3282932
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1984-04-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Suh, K; Gabel, C A; Bergmann, J E (1992) Identification of a novel mechanism for the removal of glucose residues from high mannose-type oligosaccharides. J Biol Chem 267:21671-7
Einstein, R; Gabel, C A (1991) Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization. J Cell Biol 112:81-94
Hendricks, L C; Gabel, C A; Suh, K et al. (1991) A 58-kDa resident protein of the cis Golgi cisterna is not terminally glycosylated. J Biol Chem 266:17559-65
Lazzarino, D; Gabel, C A (1990) beta-Glucuronidase is transported slowly to lysosomes in BW5147 mouse lymphoma cells: evidence that the prelysosomal enzyme is not restricted to the endoplasmic reticulum. Arch Biochem Biophys 282:100-9
Lazzarino, D; Gabel, C A (1990) Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor. J Biol Chem 265:11864-71
Borden, L A; Einstein, R; Gabel, C A et al. (1990) Acidification-dependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker. J Biol Chem 265:8497-504
O'Brien, D A; Gabel, C A; Rockett, D L et al. (1989) Receptor-mediated endocytosis and differential synthesis of mannose 6-phosphate receptors in isolated spermatogenic and sertoli cells. Endocrinology 125:2973-84
Einstein, R; Gabel, C A (1989) Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor. J Cell Biol 109:1037-46
Gabel, C A; Dubey, L; Steinberg, S P et al. (1989) Varicella-zoster virus glycoprotein oligosaccharides are phosphorylated during posttranslational maturation. J Virol 63:4264-76
Ambron, R T; Protic, J; Den, H et al. (1989) Identification of protein-bound oligosaccharides on the surface of growth cones that bind to muscle cells. J Neurobiol 20:549-68

Showing the most recent 10 out of 17 publications