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J. Biol. Chem., Vol. 281, Issue 19, 13844-13852, May 12, 2006

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Characterization of the Molecular Basis of the Drosophila Mutations in Carboxypeptidase D

EFFECT ON ENZYME ACTIVITY AND EXPRESSION^*

Galyna Sidyelyeva, Nicholas E. Baker¹, and Lloyd D. Fricker²

From the Departments of Molecular Pharmacology and Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461

Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr ^PG33 mutants do not survive past the early larval stage. These mutants have a P-element insertion within exon 1B upstream of the initiation ATG, which greatly reduces mRNA levels of all forms of CPD. Both svr ¹ and svr ^poi mutants are viable, with a silvery body color and pointed wings. The wing shape is generally similar between these two mutants, although svr ^poi mutants have smaller wings. The svr ¹ gene has a three-nucleotide deletion in exon 6, removing a leucine in a region of the protein predicted to function as a folding domain for the second CP-like domain. svr ^poi has a 1072-bp duplication of the gene that introduces a stop codon into the open reading frame, causing the truncation of the protein in the middle of the second CP-like domain. Both deletions eliminate enzyme activity of the second CP-like domain and appear to cause the misfolding of the protein. This greatly reduces the levels of the long forms of CPD protein but do not affect the levels of the short forms. Taken together, these findings suggest that lethal and viable svr alleles differ in which protein forms are affected. Flies that retain the short form are viable, whereas flies that are missing all forms of CPD do not survive past the early larval stages.

Received for publication, December 19, 2005 , and in revised form, March 21, 2006.

^* This work was primarily supported by National Institutes of Health Grants DK-51271 (to L. D. F.) and GM-61230 (to N. E. B.). The DNA sequencing facility of the Albert Einstein College of Medicine is supported in part by Cancer Center Grant CA13330. 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.

¹ Scholar of the Irma T. Hirschl Foundation for Biomedical Sciences.

² To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4225; Fax: 718-430-8954; E-mail: fricker{at}aecom.yu.edu.

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