Zhang H, Komano H, Fuller R S, Gandy S E, Frail D E
Department of Corporate Molecular Biology, Abbott Laboratories, Abbott Park, Illinois 60064.
J Biol Chem. 1994 Nov 11;269(45):27799-802.
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.
人类β-淀粉样前体蛋白（APP）是阿尔茨海默病β-淀粉样肽的跨膜前体，通过与前原α-因子融合在酿酒酵母中表达。通过对蛋白酶缺陷型酵母菌株的分析，融合蛋白经Kex2蛋白酶进行部分加工，产生全长APP，并且一部分分子在液泡中被降解。一种带有腔内而非胞质表位的可溶性APP胞外域片段被释放到培养基中，表明存在“膜蛋白溶解蛋白酶”或“分泌酶”活性的切割作用。酵母细胞含有一个C端APP片段，其迁移率与源自人类细胞中全长APP经α-分泌酶切割产生的真实C端片段相同。从酵母中免疫亲和纯化的C端APP片段的N端序列与哺乳动物和昆虫细胞中报道的序列相同。这些结果证明酵母中存在分泌酶活性。此外，这种酵母分泌酶活性可能与后生动物细胞中存在的APP加工活性有关。
