Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast.
Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.
Komano
H
H
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Seeger
M
M
Gandy
S
S
Wang
G T
GT
Krafft
G A
GA
Fuller
R S
RS
D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D016564:Amyloid beta-Protein Precursor; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human