Individual carboxypeptidase D domains have both redundant and unique functions in Drosophila development and behavior.

Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1-3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svr (PG33) mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides.

Characterization of the molecular basis of the Drosophila mutations in carboxypeptidase D. Effect on enzyme activity and expression.

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.

Characterization of Drosophila carboxypeptidase D.

Metallocarboxypeptidase D (CPD), is a 180-kDa protein that contains three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail and which functions in the processing of proteins that transit the secretory pathway. An initial report on the Drosophila melanogaster silver gene indicated a CPD-like protein with only two and a half carboxypeptidase-like domains with no transmembrane region (Settle, S. H., Jr., Green, M. M., and Burtis, K. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9470-9474). A variety of bioinformatics and experimental approaches were used to determine that the Drosophila silver gene corresponds to a CPD-like protein with three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. In addition, two alternative exons were found, which result in proteins with different carboxypeptidase-like domains, termed domains 1A and 1B. Northern blot, reverse transcriptase PCR, and sequence analysis were used to confirm the presence of the various mRNA forms. Individual domains of Drosophila CPD were expressed in insect Sf9 cells using the baculovirus expression system. Media from domain 1B- and domain 2-expressing cells showed substantial enzymatic activity, whereas medium from domain 1A-expressing cells was no different from cells infected with wild-type virus. Domains 1B and 2 were purified, and the enzymatic properties were examined. Both enzymes cleaved substrates with C-terminal Arg or Lys, but not Leu, and were inhibited by conventional metallopeptidase inhibitors and some divalent cations. Drosophila domain 1B is more active at neutral pH and greatly prefers C-terminal Arg over Lys, whereas domain 2 is more active at pH 5-6 and slightly prefers C-terminal Lys over Arg. The differences in pH optima and substrate specificity between Drosophila domains 1B and 2 are similar to the differences between duck CPD domains 1 and 2, suggesting that these properties are essential to CPD function.