Juvenile hormone binding proteins in larval fat body nuclei of Drosophila melanogaster

J. Insect Physiol.

Vol. 39,

No. 7, pp. 563-569,

0022-1910/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd

1993

Printed in Great Britain. All rights reserved

Juvenile Hormone Binding Proteins in Larval Fat Body Nuclei of Drosophila melanogaster LIRIM

SHEMSHEDINI,*,t

THOMAS

G. WILSON*,$

Received 31 March 1992; revised 7 January 1993

In previous studies juvenile hormone has been implicated as acting at the plasma membrane level or at the chromosomal level. The present study has examined juvenile hormone III biing and action in the nucleus of two Drosophilu mefanoguster tissues that respond to juvenile hormone. Juvenile hormone III was fouud to bind to nuclear preparations of larval fat body, a tissue that in adults undergoes histolysis in response to juvenile hormone. Photoafiinity labeling revealed the presence of three nuclear juvenile hormone analogue-binding proteins, two of which can be specifkally competed with juvenile hormone III. One of these proteins corresponds in size to a juvenile hormone binding protein found in greater quantity in the cytosolic fraction from several tissues of Drosophila, and the other protein is unique to the nuclear fraction. Juvenile hormone III acquired the ability to bind to DNA-ceRulose when incubated with larval fat body cytosol, but not with larval hemolymph. Moreover, juvenile hormone III was shown to stimulate RNA synthesis in male accessory glands, a tissue that responds to juvenile hormone by increased protein synthesis in several insect species. These results suggest that the juvenile hormone may act through the nucleus to effect at least part of its action. Juvenile hormone

Photoaffinity labeling Drosophila

INTRODUCTION Because juvenile hormone has important roles in insect development (Riddiford, 1985) and reproduction (Koeppe et al., 1985), much effort has been directed at determining its mode of action. One proposed mechanism is transcriptional regulation in a manner similar to that of steroid hormones (Chang et al., 1980; Engelmann, 1990). However, other studies suggest that juvenile hormone may act at the plasma membrane level to stimulate protein synthesis in male accessory glands of Drosophila and Rhodnius prolixus (Yamamoto et al., 1988; Gold and Davey, 1989). Previous work bearing on this issue has examined juvenile hormone binding proteins in various cell fractions and has led to the identification of both cytosolic (Riddiford and Mitsui, 1978; Chang et al., 1980; Koeppe et al., 1981; Roberts and Wyatt, 1983; Wisniewski and Kochman, 1984; Van Mellaert et al., 1985; Engelmann et al., 1987; Wisniewski et al., 1988; Shemshedini et al., 1990) and nuclear (Roberts and Jefferies, 1986; Engelmann et al., 1987; Palli et al., 1990) juvenile hormone binding proteins in a variety of insects. Many of these juvenile hormone *Department of Zoology, University of Vermont, Burlington, VT 05405, U.S.A. tPresent address: Laboratoire de Gknetique Moleculaire des Eucaryotes du CNRS, Unit 184 de Chimie Biologie Moleculaire et de Genie Genttique de l’fNSERM, Institut de Chimie Biologique, Facultt de Mkkcine, 1I, rue Humann, 67085 Strasbourg Cedex, France. $To whom all correspondence should be sent.

binding proteins exhibit characteristics expected of a juvenile hormone receptor, such as high binding affinity, ligand specificity, and tissue or temporal specificity. However, direct evidence for a juvenile hormone receptor is lacking. In none of these insects is the relationship between nuclear and cytosolic juvenile hormone binding proteins understood. Evidence from the vertebrate receptor literature indicates that, for at least some vertebrate steroid hormones, the receptor is found solely in the nucleus (Brenner et al., 1988; Gasc and Baulieu, 1988) while other vertebrate receptors appear to be located in both cytoplasm and nucleus. In Drosophila we have recently identified two cytosolic juvenile hormone binding proteins by photoaffinity labeling that can be competed with cold juvenile hormone III (Shemshedini et al., 1990). We now show that one, but not both, of these cytosolic juvenile hormone binding proteins also appears in nuclear fractions of larval fat body tissue. In addition, a novel smaller molecular weight (M,) juvenile hormone binding protein appears in nuclear fractions of this same tissue. MATERIALS AND METHODS Hormones

III (specific [3HIJuvenile hormone activity 11.9 Ci/mmol) was purchased from New England Nuclear while unlabeled juvenile hormone III was obtained from Sigma; both were racemic mixtures. [‘HI563

564

LIRIM SHEMSHEDINI

(1 OR,11S)-epoxyfarnesyl

diazoacetate (specific activity 14 Ci/mmol) was generously provided by Dr G. D. Prestwich, Stony Brook, NY. All juvenile hormones and analogues were stored in hexane at -20°C. Concentrations of the radiolabeled compounds were determined by radioactivity, while the concentration of unlabeled juvenile hormone III was determined by ultraviolet spectroscopy as previously described (Shemshedini et al., 1990). Animals and tissue preparation

Flies were raised at 25 + 1°C in uncrowded cultures on a cornmeal-agar-yeast-molasses diet supplemented with propionic acid to inhibit mold growth. The laboratory balancer strain First Multiple Seven (FM7), described in Lindsley and Grell (1968) was used as a methoprene-susceptible strain in this study as in previous studies (Wilson and Fabian, 1986; Shemshedini and Wilson, 1990; Shemshedini et al., 1990). These flies have a useful genetic marker and are similar to wild-type with regard to susceptibility to methoprene (Wilson and Fabian, 1986) and juvenile hormone III binding kinetics in cytosolic preparations (Shemshedini and Wilson, 1990). Third-instar larvae, collected several hours before pupariation from the walls of the culture bottles, were used for isolation of hemolymph as previous described (Shemshedini and Wilson, 1988). Hemolymph was diluted lOO-fold in Tris-EDTA buffer (Roberts and Wyatt, 1983) as previously described (Shemshedini and Wilson, 1988) before it-was used in either the hormone or DNA-cellulose binding assay. Adults were collected &4 h following eclosion and used for isolation of either larval fat body (Shemshedini and Wilson, 1990) or male accessory glands (Shemshedini et al., 1990). At this time after eclosion the larval fat body exists as separated cells but has not commenced significant autohistolysis (Postlethwait and Jones, 1978). Autohistolysis of this tissue occurs over a period of l-2 days following eclosion and is dependent on juvenile hormone (Postlethwait and Jones, 1978). Isolated larval fat body was washed 4x in TTgm buffer (Roberts and Wyatt, 1983) before being homogenized in Tris-EDTA buffer (Shemshedini and Wilson, 1990) and centrifuged at 1000 g for 10 min. The pellet was resuspended in 50 mM KPO,, pH 6.5, 8 mM MgCl,, 10 mM a-thioglycerol buffer (Roberts and Jefferies, 1986) with 1.5 M sucrose and centrifuged at 10,OOOg for 30 min, resulting in a pellet of primarily nuclei. These nuclear preparations were judged to be about 80% pure by phase-contrast microscopy. Nuclei were washed twice with Tris-EDTA buffer and resuspended in the same buffer before being used for either hormone binding or photoaffinity labeling. The supernatant from the 1000 g centrifugation was centrifuged at 10,000g for 10 min, and the resulting supernatant from this step was further centrifuged at 100,OOOg for 1.5 h, resulting in a cytosolic fraction (supernatant).

and THOMAS G. WILSON

Photoafinity labeling of cytosol and nuclei

Larval fat body cytosol and nuclei were photoaffinity labeled with [3Hlepoxyfarnesyl diazoacetate, followed by electrophoresis in a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and fluorography as previously described (Shemshedini et al., 1990). Following photolabeling, whole nuclei were solubilized in Laemmli sample buffer (Laemmli, 1970), boiled for 5 min, and subjected to electrophoresis. Hormone binding assays

Two different assays were used to measure juvenile hormone III binding by hemolymph and larval fat body cytosol or nuclei. A quantity of 0.1 ml of each tissue fraction was incubated with 70 nM [3H]juvenile hormone III in the presence (nonspecific binding) or absence (total binding) of a lo-fold excess of unlabeled juvenile hormone III for 1 h at 4°C; specific binding is defined as total minus nonspecific binding. Binding by hemolymph and cytosol was measured using the hydroxyapatite assay (Shemshedini and Wilson, 1988). Binding by nuclei was measured using the nuclear exchange assay as modified by Osir and Riddiford (1988). DNA-cellulose

binding assay

Juvenile hormone III binding to DNA-cellulose was measured using a method modified from Goidl et al. (1977). A quantity of 0.1 ml of either hemolymph or cytosolic extract from well-washed larval fat body cells was incubated with 60 nM of [3H]juvenile hormone III in the presence (nonspecific) or absence (total) of a lOO-fold excess of unlabeled juvenile hormone III for 1 h at 4°C and specific binding was determined. Afterwards, 0.2 ml of a 25% slurry of calf thymus DNA-cellulose (Sigma) in TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA) was added, vortexed vigorously, and incubated for 45 min at 4°C. The DNA-cellulose was washed by adding 1 ml of TE buffer, vortexing vigorously, and, centrifuging at 800g for 5 min. The supernatant was discarded, and the DNA-cellulose was washed five additional times with 1 ml of TE buffer each time. After the final wash, the DNA-cellulose pellet was resuspended in TE buffer and counted in Aquassure (New England Nuclear) to determine the amount of [3H] juvenile hormone III binding to DNA-cellulose. RNA and protein synthesis

Male accessory glands were dissected from 0- to 4-h-old flies as described previously (Shemshedini et al., 1990). Three pairs of glands were incubated with 5 PCi of either [3H]uridine (ICN) or [“Slmethionine (ICN) in 50 ml of (2-[N-morpholinolpropanesulfonic acid, MOPS) buffer containing 0.1 mM CaCl, (Yamamoto et al., 1988) to which different concentrations of juvenile hormone III had been added. After 3 h at 25”C, the glands were rinsed, lyophilized, and then lysed in Laemmli sample buffer. To measure RNA synthesis, contents of the lysed glands labeled with [‘Hluridine

NUCLEAR

JUVENILE

HORMONE

were transferred onto DE-81 filters (2 x 2 cm) and washed as described in Maniatis et al. (1982). The filters were counted in Aquassure to determine the amount of [‘Hluridine incorporation into RNA. Protein synthesis was measured as previously described (Shemshedini et al., 1990). To measure the effect of actinomycin D on juvenile hormone III stimulation of RNA and protein synthesis, MOPS buffer was preincubated with 1 mM juvenile hormone III and 5 M actinomycin D for 30 min at 25°C prior to addition of radiolabel and male accessory glands. The glands were then treated as described above.

BINDING

JH IIJ

565

PROTEINS

-

N

C +

-

A

_

C

+

20

66

RESULTS

Juvenile hormone binding by nuclei

In previous work we examined the cytosol of larval fat body from newly eclosed adults and found high affinity juvenile hormone binding and two juvenile hormone III-competible epoxyfarnesyl diazoacetate binding proteins (Shemshedini and Wilson, 1990). Epoxyfarnesyl diazoacetate is a photoaffinity analogue of juvenile hormone III (Prestwich et al., 1985). Since other workers have found nuclear as well as cytosolic juvenile hormone binding proteins in the same tissues of various insects (Roberts and Jefferies, 1986; Engelmann et al., 1987; Osir and Riddiford, 1988; Palli et al., 1990), we examined juvenile hormone binding in nuclei from larval fat body of Drosophila. When isolated nuclei were incubated with [3H]juvenile hormone III, one of two juvenile hormone homologues found in Drosophila (Bownes and Rembold, 1987; Sliter et al., 1987; Richard et al., 1989), specific binding of the hormone could be measured (Table 1). However, this binding by nuclei was sharply lower than that by cytosol (Table l), suggesting that if juvenile hormone III is associated with a juvenile hormone binding protein(s) in the nucleus, then the concentration of the binder in the nucleus is much lower than in the cytosol of larval fat body. Photoafinity labeling of cytosol and nuclei

To identify specific juvenile hormone binding proteins of the larval fat body cytosol and nucleus, photoaffintiy labeling was carried out using [3H]epoxyfarnesyl diazoacetate. Photolabeling of larval fat body cytosol followed by SDS-PAGE revealed a major specific binder of TABLE 1. Juvenile hormone III binding by cytosolic and nuclear preparations of larval fat body from Drosophila Cell fraction Cytosolic Nuclear

Specific binding (fmol per mg protein) 18.2 k 4.4 2.4 & 0.6

All values are means of three replicates + SEM. The mean ratios of total to nonspecific binding were cytosol, 81/21 fmol, and nuclear, 22/17 fmol.

29

FIGURE 1. SDS-PAGE of photoaffinity-labeled larval fat body nuclei (N) and cytosol (C) with [‘Hlepoxyfarnesyl diazoacetate with or without a lOO-fold excess of unlabeled juvenile hormone III. The cytosol lanes are duplicate runs to demonstrate the transient nature of the M, 50,000 band. Equivalent amounts of protein were loaded into each well. Following electrophoresis the gel was subjected to fluorography (1 month) for detection of the epoxyfarnesyl diazoacetatelabeled proteins. The approximate positions of the molecular weight standards in kDa are indicated with small arrows. Larger arrows indicate the positions of the nuclear juvenile hormone binding proteins.

[3Hlepoxyfarnesyl diazoacetate of an apparent molecular weight (M,) of 85,000 (Fig. 1); this result is similar to earlier findings (Shemshedini et al., 1990). Two other cytosolic proteins, of it4, 80,000 and 63,000, bound [3H]epoxyfarnesyl diazoacetate (Fig. l), as seen previously (Shemshedini et al., 1990). The M, 80,000 protein is a nonspecific binder (unable to be competed with juvenile hormone III), while the M, 63,000 protein is a specific binder competible by juvenile hormone III that labels at l&30% (depending on tissue preparation) of the intensity of the M, 85,000 protein (Shemshedini et al., 1990). An additional minor band of M, 50,000 is unique to one lane of this gel and is perhaps a degradative product or gel artifact; it does not appear in a duplicate lane included in Fig. 1. When whole nuclei were photolabeled, solubilized with Laemmli sample buffer, and subjected to SDS-PAGE, three major proteins of approximately equal intensity were found to bind [3H]epoxyfarnesyl diazoacetate (Fig. 1). In agreement with the hormone binding data (Table l), the labeling of the nuclear juvenile hormone binding proteins was much lower than that of the cytosolic juvenile hormone binding proteins. One of the proteins appears to be the nonspecific M, 80,000 binder. The other two proteins are competed with juvenile hormone III; one appears to be the M, 85,000

LIRIM SHEMSHEDINI

566

and THOMAS G. WILSON

cytosolic binding protein, while the other, having an M, of 30,000, is unique to the nuclear fraction. These results suggest that, based on molecular weight, the major cytosolic specific juvenile hormone binding protein is also found in the nucleus, in addition to a new juvenile hormone binding protein. Juvenile hormone binding to DNA-cellulose

If juvenile hormone acts through the nucleus, then one or both of these juvenile hormone III binding proteins in the nucleus may be a juvenile hormone receptor protein. The ability of a hormone-binding protein to bind to DNA-cellulose is a property previously used to define vertebrate steroid receptors (Kalimi et al., 1975). More recently, this property has been suggested as a characteristic of juvenile hormone receptors (Prestwich et al., 1985) and reported for a Manduca sexta nuclear juvenile hormone binding protein (Palli et al., 1990). We examined DNA-cellulose binding with Drosophila larval fat body cytosol. [3HJJuvenile hormone III showed negligible specific binding to DNA-cellulose in the absence of tissue extracts. When [3111juvenile hormone III was first incubated with larval fat body cytosol and then assayed for binding to DNA-cellulose, a high level of juvenile hormone III specific binding was found (Table 2). When the same experiment was repeated with larval hemolymph, significantly less juvenile hormone III bound to DNA-cellulose (Table 2). Larval hemolymph also contains a high affinity juvenile hormone binding protein (Shemshedini and Wilson, 1988), but its binding characteristics are very different from those of the cytosolic juvenile hormone binding protein (Shemshedini and Wilson, 1988; Shemshedini et al., 1990; unpublished). When [31-11 juvenile hormone III binding to DNA-cellulose was determined based on the amount of [3Hjjuvenile hormone III bound to protein as measured with the HAP binding assay, 75% of the protein-bound hormone in cytosolic preparations was bound to DNA-cellulose as compared to less than 5% with hemolymph (Table 2). It is clear from these results that [31-Iljuvenile hormone III has a higher affinity for DNA-cellulose when associated with cytosol than with hemolymph, suggesting that the cytosolic juvenile hormone binding protein may function in DNA binding. Juvenile hormone stimulation of RNA and protein synthesis

The presence of nuclear juvenile hormone binding proteins and the ability of [3HJjuvenile hormone III, in TABLE 2. Juvenile hormone III binding in the presence of larval fat body cytosol or larval hemolymph to DNA-cellulose Tissue Fat body cytosol Hemolymph

Specific binding (fmol per mg protein)

HAP binding (“/)

14.73 f 2.40 0.99 f 0.02

75.0 4.5

All values in fmol are means of three to four replicates f SEM.

TABLE 3. Juvenile hormone III stimulation of RNA synthesis in male accessory glands [JH III] (M) 0

10-9 10-s 10-l lo-6

RNA Synthesis (cpm) 5949&&l 9731* 1394 10,146 &-2514 10,430 k 2412 11,990* 1604

RNA synthesis is represented as cpms of [3H]uridine incorporation into RNA. All values are means of four replicates + SEM.

the presence of cytosolic extract, to bind to DNA-cellulose suggest that juvenile hormone acts in the nucleus, presumably to affect transcription. To evaluate this possibility, the effect of juvenile hormone III on RNA synthesis was studied. Although a role has been clearly demonstrated for juvenile hormone in the larval fat body autohistolysis that occurs in the first 2 days following eclosion (Postlethwait and Jones, 1978), no biochemical response to juvenile hormone has been demonstrated in this tissue. Therefore, we turned to another tissue having a known biochemical response to juvenile hormone for this evaluation. Male accessory glands have been shown to have juvenile hormoneinducible protein synthesis (Yamamoto et al., 1988; Shemshedini et al., 1990) and to contain a cytosolic juvenile hormone binding protein that, based on subunit molecular weight and juvenile hormone III binding affinity, resembles that in larval fat body (Shemshedini et al., 1990). We evaluated RNA synthesis in male accessory glands cultured in vitro in order to eliminate an indirect effect of juvenile hormone. Juvenile hormone III was found to stimulate RNA synthesis in a dose-dependent manner up to a two-fold level by the highest level of hormone tested, 10e6 M juvenile hormone III (Table 3). To determine if juvenile hormone-stimulated protein synthesis is dependent on RNA synthesis, the effect of actinomycin D on the stimulation of protein and RNA

TABLE 4. The effect of actinomycin D on juvenile hormone III stimulation of RNA and protein synthesis in male accessory glands

Condition

Response to juvenile hormone III stimulation RNA synthesis (cpm)

- Actinomycin D + Actinomycin D Condition

15,359 & 1101 4984 & 1889 Protein synthesis (x lo6 d.u.)

-Actinomycin D + Actinomycin D

1.03 f 0.26 0.95 f 0.12

RNA synthesis is represented as cpms of [‘Hluridine incorporation into RNA; protein synthesis is represented as densitometric units (d.u.) used to quantify [-”Slmethionine incorporation into protein. All values are means of three replicates + SEM.

NUCLEAR

JUVENILE

HORMONE

synthesis was studied. As Table 4 shows, actinomycin D inhibited juvenile hormone stimulation of RNA synthesis by about 70% but had no effect on protein synthesis. These results suggest that the stimulation of RNA and protein synthesis are two independent effects of juvenile hormone III in the male accessory glands. DISCUSSION The hypothesis that juvenile hormone acts in the nucleus has been proposed earlier (Chang et al., 1980; Engelmann, 1990). Since then it has received support from studies identifying nuclear juvenile hormone binding proteins with classic hormone receptor characteristics (Roberts and Jefferies, 1986; Engelmann et al., 1987; Osir and Riddiford, 1988; Palli et al., 1990). In the present work we have also found nuclear juvenile hormone binding in D. melanogaster and have identified by photoaffinity labeling two major nuclear juvenile hormone binding proteins that can be competed by juvenile hormone III. Based on electrophoretic mobility, one of these nuclear juvenile hormone binding proteins appears to be the same as the 85,000 cytosolic juvenile hormone binding protein previously identified in larval fat body cytosol (Shemshedini et al., 1990). The second nuclear juvenile hormone binding protein competible by juvenile hormone III has an M, of 30,000 and appears to be unique to the nucleus. A third nuclear juvenile hormone binding protein corresponds to a nonspecific cytosolic binder (M, 80,000) of juvenile hormone III. It seems unlikely that the presence of the 80,000 and 85,000 proteins in the nucleus is due to contamination by cytoplasm since the nuclei were washed extensively with buffer prior to photolabeling. Furthermore, novel juvenile hormone-III competible juvenile hormone binding proteins are found in the nuclear and cytoplasmic fractions: the h4, 30,000 nuclear protein and the M, 63,000 cytoplasmic protein. In addition, we have found that fractions of the cell homogenate that might contaminate a nuclear preparation (mitochondria and microsomal membranes) do not contain appreciable specific juvenile hormone III binding activity. However, the quantities of the juvenile hormone binding proteins in the nuclear fraction are considerably less than those in the cytosol, and we cannot rule out the observed activity in nuclear fractions as resulting from contaminating tightly adhering cytosolic juvenile hormone binding proteins to nuclei. The function of multiple nuclear juvenile hormone binding proteins is unclear. However, assuming that juvenile hormone is acting to regulate gene expression, it is possible that the different juvenile hormone binding proteins might be acting on different genes or sets of genes, providing another level of regulation by juvenile hormone. There is also the possibility that one of these nuclear juvenile hormone binding proteins is a juvenile hormone esterase, as it has been seen with cytosolic juvenile hormone binding proteins in the fat body of

BINDING

PROTEINS

567

locusts (Roberts and Wyatt, 1983). If, indeed, more than one nuclear juvenile hormone binding protein is functioning as a juvenile hormone receptor, then determining the functions of multiple nuclear juvenile hormone receptors should be one objective of future work on juvenile hormone receptors. Juvenile hormone III, after incubation with larval fat body cytosol, acquires the ability to bind to DNA-cellulose. Although this finding is not definitive for defining a physiological role for juvenile hormone regulation at the gene level, it does provide a preliminary suggestion of such an association. Recently, Palli et al. (1990) have shown that their nuclear juvenile hormone binding protein from Manduca binds to DNA-cellulose and to juvenile hormone-regulated endocuticle genes that have been isolated. Since no genes in Drosophila have been identified as being directly regulated by juvenile hormone, binding of Drosophila juvenile hormone binding proteins to defined DNA sequences, a more definitive experiment, must await additional work in this area. Promising results in this regard have recently appeared showing inhibition of an ecdysone-regulated heat shock protein gene by juvenile hormone III or methoprene, a juvenile hormone analogue (Berger et al., 1992). It is possible that the cytosolic protein is actually nuclear in origin and is found in the cytoplasm as an artifact of cellular fractionation, as it has been found for several vertebrate steroid receptors (Brenner et al., 1988; Gasc and Baulieu, 1988). Although this possibility cannot be ruled out at this time, the finding of juvenile hormone stimulation of protein synthesis independent of RNA synthesis in male accessory glands suggests that the cytosolic juvenile hormone binding protein indeed functions in the cytosol. Definitive answers to questions of intracellular localization of juvenile hormone receptors will be obtained only when antibodies against these proteins are available, as it has been the case for steroid hormone receptors (Brenner et al., 1988; Gasc and Baulieu, 1988). The M, 30,000 protein is similar in molecular weight to the putative juvenile hormone receptor in both Drosophila KC cells (Wang et al., 1989) and Manduca (Palli et al., 1990). Whether any similarity among the three proteins extends beyond photoaffinity labeling is unknown. Since the KC cell line is of embryonic origin, it is possible that this juvenile hormone binding protein functions as a receptor during early development and that the M, 85,000 or M, 63,000 juvenile hormone binding protein becomes important as a receptor later in development. In that case, we are perhaps detecting in our preparations the remnants of an embryonic receptor. Due to the difficulty of procurement of sufficient male accessory gland tissue, we were unable to examine nuclear preparations from this tissue. Therefore, the juvenile hormone binding proteins seen in larval fat body nuclear preparations cannot be directly compared with the RNA/protein synthesis results obtained with male accessory glands.

568

LIRIM SHEMSHEDINI

Our finding of independent juvenile hormone effects on RNA and protein synthesis in male accessory glands is in agreement with an earlier finding of copulation-induced RNA and protein synthesis in these glands (von Wyl and Chen, 1974). More recently, it has been shown that copulation increases the synthesis of rRNA and has no effect on mRNA synthesis (Schmidt et al., 1985). Therefore, if the effect of copulation on protein synthesis in male accessory glands is mediated by juvenile hormone III, as it has been suggested (Yamamoto et al., 1988), then juvenile hormone III acts on these glands to stimulate both protein and rRNA synthesis. The results obtained with actinomycin D (Table 4) also suggest that at least in male accessory glands juvenile hormone has two sites of action: cytoplasm and nucleus. Dual actions of a hormone are not unique to juvenile hormone; the vertebrate hormone, insulin, has been found to separately stimulate RNA and protein synthesis in Xenopus oocytes (Miller, 1988). Furthermore, some steroid hormones are known to have effects in the plasma membrane as well as in the nucleus (Pietras and Szego, 1975; Maller et al., 1979; Nenci et al., 1981). Our results suggest that in Drosophila larval fat body a major specific juvenile hormone binding protein is common to both the cytoplasm and the nucleus and that each of these fractions also contains unique specific juvenile hormone binding proteins. Future work with purified juvenile hormone binding proteins and antibodies to each should establish the relationship between these cytosolic and nuclear juvenile hormone binding proteins.

an d THOMAS G. WILSON Gold S. M. W. and Davey K. G. (1989) The effect of juvenile hormone on protein synthesis in the transparent accessory gland of male Rhodnius prolixus.

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Prestwich G. D., Koeppe J. K., Kovalick G. E., Brown J. J., Chang E. S. and Singh A. K. (1985) Experimental techniques for photoaffinity labeling of juvenile hormone binding proteins of insects with epoxyfarnesyl diazoacetate. Meth. Enzym. 111, 509-530. Richard D. S., Applebaum S. W., Sliter T. J., Baker F. C., Schooley D. A., Reuter C. C., Henrich V. C. and Gilbert L. I. (1989) Juvenile hormone bisepoxide biosynthesis in oirro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera. Proc. natn. Acad. Sci., U.S.A. 86, 1421-1425. Riddiford L. M. (1985) Hormone action at the cellular level. In Comprehensive

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Schmidt T., Stumm-Zollinger E. and Chen P. S. (1985) Protein metabolism of Drosophila melanogaster male accessory glands-III.

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Shemshedini L., Lanoue M. and Wilson T. G. (1990) Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster. J. biol. Chem. 265, 1913-1918. Sliter T. J., Sedlak B. J., Baker F. C. and Schooley P. A. (1987) Juvenile hormone in Drosophila melanogaster: identification and titer determination during development. Insect Biochem. 17, 161-165. Van Mellaert H., Theunis S. and DeLoof A. (1985) Juvenile hormone binding proteins in Sarcophaga bullata hemolymph and vitellogenic ovaries. Insect Biochem. 15, 655661. Wang X., Chang E. S. and O’Connor J. D. (1989) Purification of the Drosophila KCcell juvenile hormone binding protein. Insecf Biochem. 19, 327-335.

BINDING

PROTEINS

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Wilson T. G. and Fabian J. (1986) A Drosophila melanogasfer mutant resistant to a chemical analog of juvenile hormone. Devl Biol 118, 196201. Wisniewski J. R. and Kochman M. (1984) Juvenile hormone-binding protein from the silk gland of Galleria mellonella. FEBS Leu. 171, 127-130.

Wisniewski J. R., Wawrzenczyk C., Prestwich G. D. and Kochman M. (1988) Juvenile hormone binding proteins from the epidermis of Galleria mellonella. Insect Biochem. 18, 29-36.

von Wyl E. and Chen P. (1974) Paragonienproteine der Adultmlnnchen von Drosophila melanogaster : Elektrophoretisches Muster und in vitro Synthese. Revue suisse Zool. 81, 655662. Yamamoto K., Chadarevian A. and Pellegrini M. (1988) Juvenile hormone action mediated in male accessory glands of Drosophila by calcium and kinase C. Science 239. 916919.

Acknowledgements-We would like to thank Dr G. D. Prestwich for providing the photoaffinity analogue. This work was supported by a NIH grant to T.G.W.