CHARACTERIZATION OF BETA-AMYLOID PEPTIDE PRECURSOR PROCESSING BY THE YEAST YAP3 AND MKC7 PROTEASES

Two proteases, denoted beta- and gamma-secretase, process the beta-amy loid peptide precursor (APP) to yield the A beta peptides involved in Alzheimer's disease. A third protein, alpha-secretase, cleaves APP nea r the middle of the A beta sequence and thus prevents AP formation, Th ese enzymes have defied identification. Because of its similarity to t he systems of mammalian cells the yeast secretory system has provided important clues for finding mammalian processing enzymes, When express ed in Saccharomyces cerevisiae APP is processed by enzymes that posses s the specificity of the alpha-secretases of multicellular organisms. APP processing by alpha-secretases occurred in sec1 and sec7 mutants, in which transport to the cell surface or to the vacuole is blocked, b ut not in sec17 or sec18 mutants, in which transport from the endoplas mic reticulum to the Golgi is blocked. Neutralization of the vacuole b y NH4Cl did not block alpha-secretase action, The time course of proce ssing of a pro-alpha-factor leader-APP chimera showed that processing by Kex2 protease, a Golgi protease that removes the leader, preceded p rocessing by alpha-secretase. Deletions of the genes encoding the GPI- linked aspartyl proteases Yap3 and Mkc7 decreased alpha-secretase acti vity by 56 and 29%, respectively; whereas, the double deletion decreas ed the activity by 86%. An altered form of APP-695, in which glutamine replaced Lys-612 at the cleavage site, is cleaved by Yap3 at 5% the r ate of the wild-type APP. Mkc7 protease cleaved APP (K612Q) at about 2 0% the rate of wild-type APP, The simplest interpretation of these res ults is that Yap3 and Mkc7 proteases are alpha-secretases which act on APP in the late Golgi. They suggest that GPI-linked aspartyl protease s should be investigated as candidate secretases in mammalian tissues. (C) 1997 Elsevier Science B.V.