A4 (A
) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is aproteolytic product of the amyloid precursor protein (APP). Endoproteases, termed
- and
-secretase,release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage withinthe
A4
domain by
-secretase. To study the conservation of APP processing in lower eukaryotes, theyeast
Pichia pastoris was transfected with human APP
695 cDNA. In addition to the full-length integraltransmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culturemedium. Most sAPP comprised the N-terminal moiety of
A4 and corresponds to sAPP
, the productof
-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibodyspecific for sAPP
(the ecto
domain released by
-secretase cleavage). Analysis of the cell lysates withspecific antibodies also detected membrane-associated C-terminal fragments corresponding to the productsof
and
cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directedat distinct epitopes of the
A4
domain yielded a 4 kDa product with the same electrophoretic mobility as
A4 synthetic peptide. These results suggest that the
-,
-, and
-secretase cleavages are conserved inyeast and that
P. pastoris may offer an alternative to mammalian cells to identify the proteases involvedin the generation of AD
A4 amyloid.