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J. Biol. Chem., Vol. 280, Issue 30, 27481-27490, July 29, 2005

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Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme^*

Seonil Kim, Andrea N. Lapham¶, Christopher G. K. Freedman¶, Tiffany L. Reed¶, and Walter K. Schmidt¶||

From the Departments of Cellular Biology and ^¶Biochemistry and Molecular Biology, the University of Georgia, Athens, Georgia 30602

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.

Received for publication, December 17, 2004 , and in revised form, June 7, 2005.

^* This work was supported in part by funds from the University of Georgia. 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.

Supported by a University of Georgia graduate school assistantship.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 120 Green St., Athens, GA 30602. Tel.: 706-583-8241; Fax: 706-542-1738; E-mail: wschmidt{at}bmb.uga.edu.

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