Localization and processing of beta-amyloid precursor protein in the rat brain and yeast Saccharomyces cerevisiae.
Item
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Title
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Localization and processing of beta-amyloid precursor protein in the rat brain and yeast Saccharomyces cerevisiae.
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Identifier
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AAI9618122
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identifier
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9618122
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Creator
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Zhang, Wei.
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Contributor
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Adviser: David Lee Miller
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Date
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1996
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Neuroscience | Biology, Molecular
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Abstract
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Mammalian cells can metabolize transmembrane {dollar}\beta{dollar}-amyloid precursor protein (APP), which is implicated in Alzheimer's disease, in several ways that result in the secretion of the ectodomain and amyloid {dollar}\beta{dollar}-protein (A{dollar}\beta{dollar}). We have investigated the subcellular locations of APP in rat brains. Immunoisolated APP-containing vesicles (APPV) show a concomitant enrichment of APP and synaptophysin (synaptic vesicle marker); a synaptic vesicle-enriched fraction (LP2) also reveals the co-existence of the two proteins. More importantly, purified synaptic vesicles contain 10-15% of APP in the brain. Therefore, APP is a constituent of synaptic vesicles and may regulate their functions.;Yeast contains precursor-converting enzymes homologous to their mammalian counterparts, and is susceptible to genetic manipulation. As a first step toward identifying APP-processing enzymes (secretases), we have studied the processing of human APP in transformed yeast (Saccharomyces cerevisiae). The cells express high levels of APP (1% of total protein), and, like mammalian cells, yeast can cleave APP within the A{dollar}\beta{dollar} region to generate a soluble C-terminal truncated fragment (sAPP), while a complementary C-terminal fragment (CTF) accumulates in the membrane. Amino acid sequencing demonstrates that the secretory cleavage site of APP is 12 residues away from the membrane-spanning sequence and is identical to "{dollar}\alpha{dollar}-secretase site" in mammalian cells. A pulse-chase study reveals that the half-life of APP turnover is 30-40 min; the parallel appearance of sAPP and CTF further confirms that they are the products of same protease(s). Unlike its mammalian counterpart, yeast {dollar}\alpha{dollar}-secretase is basic amino acid-specific; however, processing of APP occurs efficiently in yeast mutants defective in Yap3 and Kex2 peptidases, both of which cleave at basic amino acids. Yeast {dollar}\alpha{dollar}-secretase is not a vacuolar protease because APP cleavage proceeds undiminished in both a vacuolar protease-deficient strain and cells treated with a lysosomotropic agent, NH{dollar}\sb4{dollar}Cl. Using temperature-sensitive secretion mutants, we located the yeast {dollar}\alpha{dollar}-secretase in the Golgi, possibly in a distinct late Golgi region distal to the compartment containing Kex2/Kex1. We conclude that yeast possesses a unique protease that resembles mammalian {dollar}\alpha{dollar}-secretase; therefore, yeast can serve as a model for understanding mammalian APP secretase.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
