Secretion of yeast aspartic protease 3 is regulated by its carboxy-terminal tail: characterization of secreted YAP3p.
Yeast aspartic protease 3 (YAP3p), a basic-residue specific proprotein processing enzyme, was shown to be a membrane-associated protease. The membrane association of YAP3p was demonstrated to be through a glycophosphatidylinositol anchor situated in the carboxy terminus of the enzyme. Carboxy-terminal truncation of YAP3p by 37 amino acids resulted in secretion of YAP3p into the growth medium. Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two secreted forms of YAP3p with apparent molecular masses of approximately 180 and approximately 90 kDa. YAP3p has an isoelectric point of approximately 4.5 as determined by isoelectric focusing gel electrophoresis. Treatment of YAP3p with endoglycosidase H reduced the size of both forms of the protein to approximately 65 kDa, consistent with the presence of 10 potential N-linked glycosylation sites in the deduced amino acid sequence of this protein. Removal of the N-linked sugars did not affect the enzymatic activity of YAP3p. Analysis of the effect of temperature on the stability and the rate of enzymatic activity of YAP3p showed that the enzyme retained 100% of its activity when incubated for 1 h at 37 degrees C, while incubation at 50 degrees C for 1 h resulted in approximately 80% loss of activity. The dependence of activity on temperature demonstrated a calculated Q10 of 1.95.
Cawley
N X
NX
Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wong
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M
Pu
L P
LP
Tam
W
W
Loh
Y P
YP
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043484:Proprotein Convertases; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013816:Thermodynamics
D017261:Glycosylphosphatidylinositols; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases