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J. Biol. Chem., Vol. 280, Issue 13, 12523-12535, April 1, 2005

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Functional Overexpression of -Secretase Reveals Protease-independent Trafficking Functions and a Critical Role of Lipids for Protease Activity^*

Jonathan D. J. Wrigley, Irina Schurov, Emma J. Nunn, Agnes C. L. Martin, Earl E. Clarke, Semantha Ellis, Timothy P. Bonnert, Mark S. Shearman, and Dirk Beher

From the Department of Molecular and Cellular Neuroscience, Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, United Kingdom

Presenilins appear to form the active center of -secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue -secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, -secretase appears to play a role in the trafficking of the -amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized -secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. -Secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the -secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive -secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of -secretase.

Received for publication, November 19, 2004

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a figure.

To whom correspondence should be addressed: Dept. of Molecular and Cellular Neuroscience, Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Eastwick Rd., Harlow, Essex CM20 2QR, UK. Tel.: 44-1279-440478; Fax: 44-1279-440712; E-mail: dirk_beher{at}merck.com.

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