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.
Department of Corporate Molecular Biology, Abbott Laboratories, Abbott Park, Illinois 60064.
H
H
Zhang
H
H
Komano
R S
RS
Fuller
S E
SE
Gandy
D E
DE
Frail
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