Human β-secretase activity in yeast detected by a novel cellular growth selection system

• Published in: Biochimica et biophysica acta Vol. 1620; no. 1; pp. 167 - 178
• Main Authors: Lüthi, Urs, Schaerer-Brodbeck, Claudia, Tanner, Stefan, Middendorp, Oliver, Edler, Karin, Barberis, Alcide
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 17.03.2003
• Subjects: Alzheimer; Amyloid precursor protein; Amyloid Precursor Protein Secretases; Apolipoproteins - chemistry; APP; Aspartic Acid Endopeptidases - analysis; Aspartic Acid Endopeptidases - antagonists & inhibitors; Aspartic Acid Endopeptidases - chemistry; BACE; beta-Fructofuranosidase; Cell Membrane - enzymology; Cytoplasm - enzymology; Endopeptidases; Enzyme Activation; Glycoside Hydrolases - chemistry; Humans; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Serum Amyloid A Protein - chemistry; Substrate Specificity; β-secretase; β-secretase; APP; Alzheimer; Saccharomyces cerevisiae; Amyloid precursor protein; BACE
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(02)00529-9

Sequential processing of the transmembrane amyloid precursor protein (APP) by the β-secretase BACE and by the γ-secretase causes secretion of Aβ peptides. Extracellular aggregation of these peptides in the brain is a major hallmark of Alzheimer's disease. For therapeutic purposes and the development of specific inhibitors, it is important to characterize these secretases. We have established a cellular growth selection system for functional expression of human BACE in the yeast Saccharomyces cerevisiae. A fragment of APP bearing the β-site, the transmembrane domain and the cytosolic tail was fused to the C-terminus of the yeast enzyme invertase, which is normally secreted to allow cell growth in the presence of sucrose as the sole carbon source. The resulting invertase–APP fusion protein was expressed as a type-I transmembrane protein in intracellular compartments of yeast cells lacking endogenous invertase. In these cells, co-expression of human BACE restored cell growth on selective plates upon cleavage of the invertase–APP fusion protein. The cellular growth selection system presented here can be generally applied to screen for secretases that specifically cleave membrane-bound substrates. Furthermore, this system provides the basis for a high-throughput screen for identifying secretase inhibitors that are active in eukaryotic cells.