Purification and characterization of a paired basic residue-specific yeast aspartic protease encoded by the YAP3 gene. Similarity to the mammalian pro-opiomelanocortin-converting enzyme.
Yeast cells express an alternate enzyme encoded by the YAP3 gene which can process pro-alpha-mating factor when this pheromone is overexpressed in KEX2-deficient mutants. The YAP3 gene product is an aspartic protease (YAP3) that cleaves at paired basic residues (Egel-Mittani, M., Flygenring, H.P., and Hansen, M. T. (1990) Yeast 6, 127-137). In this study, the YAP3 gene was overexpressed in the BJ 3501 strain of Saccharomyces cerevisiae. YAP3 was purified to apparent homogeneity using concanavalin A and pepstatin A affinity chromatography. The enzyme was characterized as an M(r) 68,000 glycoprotein with a pH optimum of 4.0-4.5. It was inhibited by pepstatin A and activated by 5 mM Ca2+. YAP3 cleaved at paired basic residues of mouse pro-opiomelanocortin (POMC) to yield adrenocorticotropin (ACTH) and beta-lipotropin (LPH); human beta-LPH to yield beta-endorphin-(1-31), beta-endorphin-(1-29), beta-endorphin-(1-28), gamma-LPH, and beta-melanocyte-stimulating hormone; and bovine N-POMC1-77 to yield gamma 3-melanocyte-stimulating hormone. It also cleaved the tetrabasic residues of ACTH1-39 to yield primarily ACTH1-15 and Lys-Arg-corticotropin-like intermediate lobe peptide. The physical properties, pH optimum, and specificity of YAP3 indicate that it is a homologue of the mammalian POMC-converting enzyme (EC 3.4.23.17), a paired basic residue-specific aspartic protease from bovine pituitary intermediate lobe secretory granules (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205).
Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
A V
AV
Azaryan
M
M
Wong
T C
TC
Friedman
N X
NX
Cawley
F E
FE
Estivariz
H C
HC
Chen
Y P
YP
Loh
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism*; D005800:Genes, Fungal; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D001615:beta-Endorphin / Q000378:metabolism; D008083:beta-Lipotropin / Q000378:metabolism
D009842:Oligopeptides; D011480:Protease Inhibitors; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D008083:beta-Lipotropin; D010450:Endopeptidases; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases