Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta

A putative juvenile hormone esterase (JHE) binding protein, P29, was isolated from the tobacco hornworm Manduca sexta [J. Biol. Chem. 275(3), 1802-1806]. A homolog of P29 was identified in Drosophila melanogaster by sequence alignment. This gene, CG3776 was cloned, recombinant DmP29 expressed in Escheriscia coli and two anti-DmP29 antisera raised. In vitro binding of the P29 homolog to Drosophila JHE was confirmed. P29 mRNA and an immunoreactive protein of 25 kDa were detected in Drosophila larvae, pupae and adults. The predicted size of the protein is 30 kDa. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6 kDa N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25 kDa protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five cells showed that the N-terminal targeting sequence is shorter than predicted, and that a second, internal mitochondrial targeting signal is also present. An immunoreactive protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29. The function of P29 in mitochondria and the possible interaction with JHE are discussed.     

Only one esterase of Drosophila melanogaster is likely to degrade juvenile hormone in vivo

Previously we identified juvenile hormone esterase (JHE) from Drosophila melanogaster by the criteria that it showed both appropriate developmental expression and kinetics for juvenile hormone (JH). We also noted three further esterases of D. melanogaster with some JHE-like characteristics, such as a GQSAG active site motif, a particular amphipathic helix, or close phylogenetic relationship with other JHEs. In this study, these JHE-like enzymes were expressed in vitro and their kinetic parameters compared with those of the previously identified JHE. Despite considerable phylogenetic distance between some of the esterases, they could all hydrolyse racemic JHIII. However, only the previously identified JHE had kinetic parameters (K(M) and k(cat)) towards various forms of JH (racemic or individual isomers of JHIII, JHII, JHI, and methyl farnesoate) consistent with a physiological role in JH regulation. Furthermore, only this JHE showed a preference for artificial substrates with acyl chain lengths similar to that of JH. This suggests that there is probably only one physiologically functional JHE in D. melanogaster but multiple esterases with JH esterase activity. Genomic comparisons of the selective JHE across 11 other Drosophila species showed a single orthologue in 10 of them but Drosophila willistoni has 16 full-length copies, five of them with the GQSAG motif and amphipathic helix.     

Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster

The larval fat body of newly eclosed adults of Drosophila melanogaster was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein we have identified mediates this JH effect in male accessory glands and thus is acting as a JH receptor.     

Purification of the Drosophila Kc cell juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from the cytosol of Drosophila melanogaster Kc cells has been purified with the use of a juvenile hormone photoaffinity analog, 10,11-epoxy (2E, 6E) farnesyl diazoacetate (EFDA). The purification procedure consists of five chromatographic steps and the end product of the purification procedure showed homogeneity by means of both native and SDS polyacrylamide gel electrophoresis. Furthermore, using a racemic mixture of the natural hormone, [³H]juvenile hormone III (JH III), as the radioligand in this purification procedure, we demonstrate that the purified protein is likely the authentic intracellular JHBP.     

LUMP is a putative double-stranded RNA binding protein required for male fertility in Drosophila melanogaster

In animals, male fertility requires the successful development of motile sperm. During Drosophila melanogaster spermatogenesis, 64 interconnected spermatids descended from a single germline stem cell are resolved into motile sperm in a process termed individualization. Here we identify a putative double-stranded RNA binding protein LUMP that is required for male fertility. lump(1) mutants are male-sterile and lack motile sperm due to defects in sperm individualization. We show that one dsRNA binding domains (dsRBD) is essential for LUMP function in male fertility. These findings reveal LUMP is a novel factor required for late stages of male germline differentiation.     

Stress-reactivity of a Drosophila melanogaster strain with impaired juvenile hormone action

Met(27) is a null allele of the Methoprene-tolerant gene of D. melanogaster that shows resistance to the toxic effects of both juvenile hormone (JH) and a JH analog, methoprene. The mechanism of resistance appears to be altered JH reception. We measured fertility, JH-hydrolyzing activity, and dopamine (DA) levels in Met(27) and Met(+) flies under normal (25 degrees C) and heat-stress (38 degrees C) conditions. We show that under normal conditions Met(27) females have JH-hydrolyzing activity and fertility lower than Met(+), but DA content did not differ between the two strains. At 38 degrees C Met(27) flies show no impairment in JH-hydrolyzing activity in response to stress, but they do show lower DA levels and impaired reproduction. The results with Met(27) are consistent with the previous hypothesis that the alteration in fertility that follows heat stress in D. melanogaster could result from alteration in the JH endocrine system.     

A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone

Methoprene, a chemical analog of juvenile hormone, is toxic when applied to late third-instar larvae of Drosophila melanogaster. Using an ethyl methane sulfonate mutagenesis screen, we have selected two noncomplementing mutants, one of which is nearly 100 times more resistant than wild-type to either methoprene or juvenile hormone III topically applied or incorporated into the diet. The mutation, named methoprene-tolerant (Met), also confers resistance to methoprene-induced pseudotumor formation in larvae as well as to juvenile hormone III- or methoprene-induced vitellogenic oocyte development in adult females. Met adults show little or no cross-resistance to four other insecticides. The mutation was mapped by recombination to a location 35.4 on the X-chromosome and uncovered by chromosomes deficient for the region 10C2-10D4. Complementation was observed between Met and a lethal allele of the RNA polymerase II locus, which is also found in this region. Since the Met mutation also confers resistance to methoprene-induced abnormalities in adult cuticle formation, the autonomy of Met expression could be evaluated in flies mosiac for this mutation. Autonomous expression of Met was found both in abdominal cuticle as well as in external male genitalia. The characteristics of Met are consistent with those expected of a mutant having altered juvenile hormone reception in target tissue.     

Juvenile hormone esterases of Lepidoptera II. Isoelectric points and binding affinities of hemolymph juvenile hormone esterase and binding protein activities

Summary The juvenile hormone esterase (JHE) and juvenile hormone binding protein (JHBP) activities from the last larval instar of 14 species of Lepidoptera (Pieris rapae, Colias eurytheme, Danaus plexippus, Junonia coenia, Hemileuca nevadensis, Pectinophora gossypiella, Spodoptera exigua, Trichoplusia ni, Heliothis virescens, Orygia vetusta, Ephestia elutella, Galleria mellonella, Manduca sexta andEstigmene acrea) were analyzed by analytical isoelectric focusing (IEF). While the multiplicity and isoelectric point of these proteins varied, all of them were mildly acidic (pI 4.0–7.0), and a large number of the species possessed only a single JHE and/or JHBP activity. The Michaelis constants (K _m's) of the whole hemolymph JHE activities from selected species for JH III were in the range of 10^–7M. The equilibrium dissociation constantK _d of the JHBP was determined by Scatchard analysis for selected species as well, with the majority of species having aK _d near 10^–7M. This information is consistent with JHE acting as a scavenger for JH at various times during development and relying entirely on mass action to remove JH from its protective JHBP complexes. The JHBP should limit nonspecific binding and thus facilitate the rapid transport of the intact hormone through-out the hemocoel. These data indicate that the species currently used in the study of the developmental biology of the Lepidoptera are biochemically similar to a variety of other species in this order.Abbreviations JH juvenile hormone - JHE juvenile hormone esterase - JHBP juvenile hormone binding protein - IEF isoelectric focusing - EPPAT O-ethyl-S-phenyl phosphoramidothiolate - DFP O O-diisopropyl phosphofluoridate     

P-M hybrid dysgenesis affects juvenile hormone metabolism in Drosophila melanogaster females

This paper studies the metabolism of the juvenile hormone, which affects gonads functioning in Drosophila melanogaster females under P-M hybrid dysgenesis. It is shown that dysgenic females grown at 29°C have increased levels of the juvenile hormone (its degradation and stress reactivity are reduced), which apparently is a compensatory response to ovarian hypoplasia.     

Stress-reactivity and juvenile hormone degradation in Drosophila melanogaster strains having stress-related mutations

Juvenile hormone (JH) degradation was studied under normal and stress conditions in young and matured females of Drosophila melanogaster strains having mutations in different genes involved in responses to stress It was shown that (1) the impairment in heat shock response elicits an alteration in stress-reactivity of the JH system; (2) the impairment JH reception causes a decrease of JH-hydrolysing activity and of stress-reactivity in young females, while in mature ones stress reactivity is completely absent; (3) the absence of octopamine results in higher JH-hydrolysis level under normal conditions and altered JH stress-reactivity; (4) the higher dopamine content elicits a dramatic decrease of JH degradation under normal conditions and of JH stress-reactivity. Thus, the impairments in any component of the Drosophila stress reaction result in changes in the reponse of JH degradation system to stress. The role of JH in the development of the insect stress reaction is discussed.     

In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster

Summary Pinocytotic activity has been analyzed in Drosophila oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm.In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone.     

Unfolding and refolding of juvenile hormone binding protein

Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements, and spectroscopic methods. Molecules of JHBP change their conformation from a native state via two unstable intermediates to a denatured state. The first intermediate appears in a compact state, because it slightly changes its molecular size and preserves most of the JHBP secondary structure of the native state. Although the second intermediate also preserves a substantial part of the secondary structure, it undergoes a change into a noncompact state changing its Stokes radius from approximately 30 to 39 A. Refolding experiments showed that JHBP molecules recover their full protein structure, as judged from the CD spectrum, fluorescence experiments, and JH binding activity measurements. The free energy of unfolding in the absence of the denaturant, DeltaG(D-N), is calculated to be 4.1 kcal mol(-1).     

Biochemical properties of esterase 6 in Drosophila melanogaster

Biochemical properties of esterase 6 in Drosophila melanogaster were investigated using partially purified preparations from three genotypes, 1/1, 1/2, and 2/2. The molecular weight of the enzyme is estimated to be about 90,000, and treatment with sodium dodecylsulfate cleaves the enzyme into four units with a molecular weight of about 22,000. The activity toward 28 naturally occurring esters was assayed and shown to vary considerably with substrate, the 1/1 preparation having in general higher activity than 1/2 and 2/2, which were very similar. Heat sensitivity, the effect of metal ions, and the effects of the presence or absence of an end product were also studied. The differences demonstrated between allozymes would allow considerable scope, under appropriate conditions, for differential selection to operate between genotypes.Supported in part by an SRC Research Studentship (N.D.D.).     

Overexpression of Methoprene-tolerant, a Drosophila melanogaster gene that is critical for juvenile hormone action and insecticide resistance

The Methoprene-tolerant (Met) gene of Drosophila melanogaster is involved in both juvenile hormone (JH) action and resistance to JH insecticides, such as methoprene. Although the consequences of Met mutations on development and methoprene resistance are known, no studies have examined Met+ overexpression. Met+ was overexpressed in transgenic lines with various promoters that drive overexpression to different levels. Flies expressing either genomic or cDNA Met+ transgenes showed higher susceptibility to both the morphogenetic and toxic effects of methoprene, consistent with the hormone-binding property of MET. Both the sensitive period and lethal period were the same as seen for non-overexpressing Met+ flies. However, continual exposure of high-overexpressing Met+ larvae to borderline-toxic or higher methoprene doses advanced the sensitive period from prepupae to first instar and the lethal period from pharate adults to larvae and early pupae. When expression of transgenic UAS-Met+ was driven to high levels by either an actin-GAL4 or tubulin-GAL4 promoter, larvae showed high mortality in the absence of methoprene, indicating that high MET titer is lethal, perhaps resulting from expression in an inappropriate tissue. Adults overexpressing Met+ did not show enhanced oogenesis, ruling out MET as a limiting factor for this hormone-driven physiology.     

Prepupal regulation of juvenile hormone esterase through direct induction by juvenile hormone

The regulation of the prepupal peak of juvenile hormorne esterase activity was investigated and found to be directly induced by juvenile hormone. Allatectomy and reimplanation as well as juvenile hormone application experiments all indicated that the appearance of prepupal juvenile hormone esterase activity was in response to a prepupal burst of juvenile hormone. Implantation experiments indicated that the effect of juvenile hormone is not mediated through the isolated brain or subesophageal ganglion.