Proteolytic processing and secretion of human beta-amyloid precursor protein in yeast. Evidence for a yeast secretase activity.
Human beta-amyloid precursor protein (APP), the transmembrane precursor of the Alzheimer's disease beta-amyloid peptide, was expressed in the yeast Saccharomyces cerevisiae by fusion to prepro-alpha-factor. From analysis of protease-deficient yeast strains, the fusion protein underwent partial processing by Kex2 protease to generate full-length APP and a fraction of the molecules were degraded in the vacuole. A soluble APP ectodomain fragment bearing lumenal but not cytosolic epitopes was released into the media, indicating cleavage by a "membrane protein-solubilizing proteinase" or "secretase" activity. Yeast cells contained a C-terminal APP fragment that co-migrated with authentic C-terminal fragment derived from alpha-secretase cleavage of full-length APP in human cells. The N-terminal sequence of immunoaffinity purified C-terminal APP fragment from yeast was identical to that reported in mammalian and insect cells. These results demonstrate the existence of a secretase activity in yeast. Furthermore, this yeast secretase activity may be related to an APP processing activity present in metazoan cells.
Zhang
H
H
Department of Corporate Molecular Biology, Abbott Laboratories, Abbott Park, Illinois 60064.
Komano
H
H
Fuller
R S
RS
Gandy
S E
SE
Frail
D E
DE
D000544:Alzheimer Disease / Q000378:metabolism; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human