Yeast aspartic protease 3 is sorted to secretory granules and activated to process proopiomelanocortin in PC12 cells. 1996

D R Cool, and D Y Louie, and Y P Loh
Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated. The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells. Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites. This pattern of immunostaining indicates localization of YAP3p in secretory granules. Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone. However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro. Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules. Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner. This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells.

UI MeSH Term Description Entries
D007150 Immunohistochemistry Histochemical localization of immunoreactive substances using labeled antibodies as reagents. Immunocytochemistry,Immunogold Techniques,Immunogold-Silver Techniques,Immunohistocytochemistry,Immunolabeling Techniques,Immunogold Technics,Immunogold-Silver Technics,Immunolabeling Technics,Immunogold Silver Technics,Immunogold Silver Techniques,Immunogold Technic,Immunogold Technique,Immunogold-Silver Technic,Immunogold-Silver Technique,Immunolabeling Technic,Immunolabeling Technique,Technic, Immunogold,Technic, Immunogold-Silver,Technic, Immunolabeling,Technics, Immunogold,Technics, Immunogold-Silver,Technics, Immunolabeling,Technique, Immunogold,Technique, Immunogold-Silver,Technique, Immunolabeling,Techniques, Immunogold,Techniques, Immunogold-Silver,Techniques, Immunolabeling
D011333 Pro-Opiomelanocortin A 30-kDa protein synthesized primarily in the ANTERIOR PITUITARY GLAND and the HYPOTHALAMUS. It is also found in the skin and other peripheral tissues. Depending on species and tissues, POMC is cleaved by PROHORMONE CONVERTASES yielding various active peptides including ACTH; BETA-LIPOTROPIN; ENDORPHINS; MELANOCYTE-STIMULATING HORMONES; and others (GAMMA-LPH; CORTICOTROPIN-LIKE INTERMEDIATE LOBE PEPTIDE; N-terminal peptide of POMC or NPP). POMC,Pro-Opiocortin,ACTH-Endorphin Precursor,ACTH-beta-Lipotropin Precursor,Corticotropin-beta-Lipotropin Precursor,Endorphin-ACTH Precursor,Opiocortin,Pre-POMC,Pre-pro-opiocortin,Preproopiomelanocortin,Pro-ACTH-Endorphin,Pro-Opio-Melanocortin,Proopiocortin,Proopiomelanocortin,ACTH Endorphin Precursor,ACTH beta Lipotropin Precursor,Corticotropin beta Lipotropin Precursor,Endorphin ACTH Precursor,Pre POMC,Pre pro opiocortin,Pro ACTH Endorphin,Pro Opio Melanocortin,Pro Opiocortin,Pro Opiomelanocortin
D011499 Protein Processing, Post-Translational Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility. Amino Acid Modification, Post-Translational,Post-Translational Modification,Post-Translational Protein Modification,Posttranslational Modification,Protein Modification, Post-Translational,Amino Acid Modification, Posttranslational,Post-Translational Amino Acid Modification,Post-Translational Modifications,Post-Translational Protein Processing,Posttranslational Amino Acid Modification,Posttranslational Modifications,Posttranslational Protein Processing,Protein Processing, Post Translational,Protein Processing, Posttranslational,Amino Acid Modification, Post Translational,Modification, Post-Translational,Modification, Post-Translational Protein,Modification, Posttranslational,Modifications, Post-Translational,Modifications, Post-Translational Protein,Modifications, Posttranslational,Post Translational Amino Acid Modification,Post Translational Modification,Post Translational Modifications,Post Translational Protein Modification,Post Translational Protein Processing,Post-Translational Protein Modifications,Processing, Post-Translational Protein,Processing, Posttranslational Protein,Protein Modification, Post Translational,Protein Modifications, Post-Translational
D011863 Radioimmunoassay Classic quantitative assay for detection of antigen-antibody reactions using a radioactively labeled substance (radioligand) either directly or indirectly to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Non-immunogenic substances (e.g., haptens) can be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. Radioimmunoassays
D002417 Cattle Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor. Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D003594 Cytoplasmic Granules Condensed areas of cellular material that may be bounded by a membrane. Cytoplasmic Granule,Granule, Cytoplasmic,Granules, Cytoplasmic
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013997 Time Factors Elements of limited time intervals, contributing to particular results or situations. Time Series,Factor, Time,Time Factor
D014162 Transfection The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES. Transfections

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