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

Abstract

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.

Fig. 1

Exon/intron organization of the CPD gene, alternative splicing, and transgenic CPD forms created for the present study. a The gene organization and splice forms were previously determined [20] and consists of two alternative first exons that either encode an active CP domain (1B) or inactive CP-like domain (1A), alternative splicing of the intron after exon 4, which produces either short forms containing a single CP domain or longer forms containing three CP domains, and alternative splicing of the intron after exon 8, which affects the cytosolic tail. b Constructs generated for the present study and the corresponding name of the endogenous form (if such a form exists). All transgenes are under the control of the UAS promoter (arrow). 1A, inactive CP domain 1A; 1B, active CP domain 1B; 3, inactive CP domain 3; TM, transmembrane domain; tail-1 or -2, cytosolic CPD tail

Fig. 2

Effect of the various CPD forms on Drosophila survival. a Percentage of the Drosophila embryo surviving to the adult stage, normalized to wild type. b Percentage of Drosophila pupa surviving to the adult stage, normalized to wild type. The error bars show standard error of the mean (n = 3). *p < 0.05, relative to wild-type pupa survival, using Student’s t test

Fig. 3

Wing-shape rescue summary with transgenic CPD forms 2-3-t2, 1B-short, and 1B-2-3-t2. The relative length of each wing segment was normalized to the length of the corresponding segment of wild-type wing. The error bars represent standard error of the mean (n = 6). *p < 0.05; **p < 0.01 relative to wild-type wings, using Student’s t test. acv anterior cross-vein, dL distal segment of longitudinal vein, E2 wing edge between the second and third longitudinal veins, E3 wing edge between the third and fourth longitudinal veins, E4 wing edge between the fourth and fifth longitudinal veins, E5 wing edge between the fifth longitudinal vein and the alula, ml middle segment of longitudinal vein, pcv posterior cross-vein, pL proximal segment of longitudinal vein

Fig. 4

Effect of the cold exposure (5–6°C) on viable svr and transgenic CPD mutants. a Recovery from cold-induced sleep of svr ¹ and svr ^poi mutants. b Recovery from cold-induced sleep of transgenic CPD mutants 2-3-t2, 1B-short, and 1B-2-3-t2 treated for 5–7 days at 18°C to decrease the svr transgene expression. Percentage of animals awake at each time point after cold exposure (5–6°C for 4 h for viable svr mutants and 6 h for transgenic mutants) was determined using 20–25 animals of each group. The error bars represent standard error of the mean for three groups of animals of each genotype. *p < 0.05, relative to wild-type flies, using Student’s t test

Fig. 5

Effect of the ethanol vapor exposure on Drosophila viable and transgenic CPD mutants. a Ethanol intoxication of svr ¹ and svr ^poi viable mutants, backcrossed to wild-type flies. b Ethanol intoxication of transgenic CPD mutants 2-3-t2, 1B-short, and 1B-2-3-t2 reared at 18°C to decrease the svr transgene expression for 5–7 days after collection at 25°C. Percentage of animals knocked down by ethanol intoxication at each time point. The error bars represent standard error of the mean for three groups of animals (20–25 flies/group) of each genotype. *p < 0.05, relative to wild-type flies, using Student’s t test

Fig. 6

Analysis of short- and long-term memory in svr and transgenic CPD mutants, using an assay based on courtship behavior. The percentage of time a male spends engaged in courtship during a test period of 10 min is referred to as the courtship index (CI) [34]. a Wild-type, svr ^poi, and svr ¹ mutants 0-min courtship levels and 60-min courtship levels are significantly suppressed (**p < 0.001, ***p < 0.0005) compared to naive courtship levels. Courtship levels are compared within genotype; the similar changes indicate that immediate recall memory and short-term memory at 0 and 60 min remain intact in the mutant flies. For naive courtship, wild-type flies n = 16, svr ^poi n = 19, svr ¹ n = 18; 0-min memory wild-type flies n = 19, svr ^poi n = 19, svr ¹ n = 20; for 60-min memory wild-type flies n = 19, svr ^poi n = 20, svr ¹ n = 20. b Comparison of long-term memory (LTM)-trained and naive-trained courtship activity. Wild-type flies show a reduction in courtship index in the LTM-trained group, relative to the naive-trained group, indicating long-term memory. In contrast, the courtship index of the svr ^poi and svr ¹ flies is comparable between the LTM-trained and naive-trained groups, indicating no long-term memory. The LTM-trained courtship levels for the wild-type, 2-3-t2, and 1B-short groups of flies are significantly suppressed compared to naive-trained courtship levels (*p < 0.05), indicating that LTM remains intact for 2-3-t2 and 1B-short transgenic CPD mutants. Wild-type flies n = 37 for naive- and LTM-trained; svr ^poi n = 26 and 23; svr ¹ n = 26 and 20; 2-3-t2 n = 19 and 16, respectively, and for 1B-short flies n = 17

Fig. 7

Mass spectrometric quantification of AKH and processing intermediates in the corpora cardiaca of transgenic CPD mutants. Left side (shaded) adult male flies, right side adult female flies. UAS:svr was used as control. Levels of AKH-Gly-Lys-Arg (top panel), AKH-Gly-Lys (middle panel), and AKH (bottom panel), measured as intensity ratio using heavy isotope-labeled AKH* as internal standard. n is shown in brackets in c. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant (Mann–Whitney test)

Fig. 8

Mass spectrometric quantification of AKH and processing intermediates in the corpora cardiaca of flies with AKH-GAL4-driven overexpression of CPD domains. Left side (shaded) adult male flies, right side adult female flies. The AKH-GAL4 parental line was used as control. Levels of AKH-Gly-Lys-Arg (top panel), AKH-Gly-Lys (middle panel), and AKH (bottom panel) measured as intensity ratio using heavy isotope-labeled AKH* as internal standard. n is shown in brackets in c. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant (Mann–Whitney test)