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Subcellular location and function of a putative juvenile hormone esterase binding protein in Drosophila melanogaster

Dissertation
Author: Zhiyan Liu
Abstract:
Insect development, metamorphosis and reproduction are regulated in part by the action of juvenile hormone (JH). The titer of JH is regulated in turn by the action of the enzymes juvenile hormone epoxide hydrolase and juvenile hormone esterase (JHE). Because of the potential for disruption of regulation of insect development through perturbation of the action of JH, the biology of JHE has been well studied. A putative juvenile hormone esterase binding protein, P29 was identified in the tobacco hornworm, Manduca sexta. Following sequencing of the Drosophila melanogaster genome, we identified a homolog of P29 in D. melanogaster, and used this insect for analysis of the biology and function of P29 in relation to JHE. The gene encoding D. melanogaster P29 (DmP29), CG3776 was cloned, recombinant DmP29 expressed in E. 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 30kD. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6kD N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25kD protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five(TM) 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. We investigated the potential ligands of DmP29 by testing three hypotheses: (i) DmP29 binds to D. melanogaster JHE: We produced a stably transformed insect cell line that expresses DmJHE and confirmed that DmP29 binds to D. melanogaster P29. DmJHE binds to both the 25 kD and 50 kD immunoreactive proteins. (ii) DmP29 binds other, non-specific esterases including two esterases predicted to be targeted to the mitochondria: We did not detect any interaction between DmP29 and non-specific esterases. (iii) DmP29 binds to other proteins in D. melanogaster : Ligand blot analysis, immunoprecipitation experiments and affinity binding experiments showed that larval serum protein 1 binds the 25 kD P29. The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolysed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant, given the high abundance, and "sticky" nature of these proteins. Interaction of DmP29 with LSP may result from non-specific associations. We used P29 hypo- and hyper-expression mutants to elucidate the function of P29 and the potential interaction of P29 with JHE. The hypomorphic mutant EP835 of P29 had reduced JHE activity when compared to wild type flies. Hyperexpression of P29 in EP/Gal4 during the early larval stages was lethal, while hyperexpression during the third instar resulted in reduced size of adult flies. This phenotype showed that overexpression of P29 interfered with insect development. Hyperexpression in newly eclosed but not in older females resulted in reduced fecundity, indicating that overexpression of P29 affected ovarian development. Fecundity was not affected by P29 hyperexpression in the male. Hypermorphic adults exhibited male-male courtship behavior. Hyperexpressed females showed reduced receptivity to males. Hyperexpressed females had decreased production of courtship pheromone, cis, cis-7, 11-hepta cosadiene, which resulted in male flies being unable to locate female flies. Hyperexpression of P29 in males resulted in decreased production of the aggregation pheromone, cis-vaccenyl acetate. For EP835/Gal4, the hypermorphic mutant, all hyperexpression phenotypes were consistent with a reduced JH titer in Drosophila. Flies that hypo- or hyper-expressed P29 had a significantly shorter lifespan: Reduced lifespan correlated with increased egg production (hypomorphic flies) and hyperactivity (hypermorphic flies), respectively. Hence, the titer of P29 appeared to be positively correlated with the titer of JHE and negatively correlated with the titer of JH. Based on the collective phenotypes and detection of JHE activity in mitochondria, we hypothesize that JHE is stored in mitochondria and that P29 functions in transport of JHE to the cytosol.

ii TABLE OF CONTENTS

ABSTRACT iii CHAPTER 1. General introduction 1 CHAPTER 2. Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta 39 CHAPTER 3. Ligands of the putative juvenile hormone esterase binding protein in Drosophila melanogaster 69

CHAPTER 4. Misexpression of a putative juvenile hormone esterase binding protein in Drosophila melanogaster 104

CHAPTER 5. General conclusions 141 ACKNOWLEDGEMENTS 149 APPENDIX 1. Expression of the mitochondrial non-specific esterases α-E1 and cricklet in insect cells 150

APPENDIX 2. Null mutant generation 154

iii ABSTRACT

Insect development, metamorphosis and reproduction are regulated in part by the action of juvenile hormone (JH). The titer of JH is regulated in turn by the action of the enzymes juvenile hormone epoxide hydrolase and juvenile hormone esterase (JHE). Because of the potential for disruption of regulation of insect development through perturbation of the action of JH, the biology of JHE has been well studied. A putative juvenile hormone esterase binding protein, P29 was identified in the tobacco hormworm, Manduca sexta. Following sequencing of the Drosophila melanogaster genome, we identified a homolog of P29 in D. melanogaster, and used this insect for analysis of the biology and function of P29 in relation to JHE.

The gene encoding D. melanogaster P29 (DmP29), CG3776 was cloned, recombinant DmP29 expressed in E. 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 30kD. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6kD N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25kD protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five TM

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

iv protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29.

We investigated the potential ligands of DmP29 by testing three hypotheses: (i) DmP29 binds to D. melanogaster JHE: We produced a stably transformed insect cell line that expresses DmJHE and confirmed that DmP29 binds to D. melanogaster P29. DmJHE binds to both the 25 kD and 50 kD immunoreactive proteins. (ii) DmP29 binds other, non-specific esterases including two esterases predicted to be targeted to the mitochondria: We did not detect any interaction between DmP29 and non-specific esterases. (iii) DmP29 binds to other proteins in D. melanogaster: Ligand blot analysis, immunoprecipitation experiments and affinity binding experiments showed that larval serum protein 1 binds the 25 kD P29. The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolysed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant, given the high abundance, and “sticky” nature of these proteins. Interaction of DmP29 with LSP may result from non-specific associations.

We used P29 hypo- and hyper-expression mutants to elucidate the function of P29 and the potential interaction of P29 with JHE. The hypomorphic mutant EP835 of P29 had reduced JHE activity when compared to wild type flies. Hyperexpression of P29 in EP/Gal4 during the early larval stages was lethal, while hyperexpression during the third

v instar resulted in reduced size of adult flies. This phenotype showed that overexpression of P29 interfered with insect development. Hyperexpression in newly eclosed but not in older females resulted in reduced fecundity, indicating that overexpression of P29 affected ovarian development. Fecundity was not affected by P29 hyperexpression in the male. Hypermorphic adults exhibited male-male courtship behavior. Hyperexpressed females showed reduced receptivity to males. Hyperexpressed females had decreased production of courtship pheromone, cis, cis-7, 11-hepta cosadiene, which resulted in male flies being unable to locate female flies. Hyperexpression of P29 in males resulted in decreased production of the aggregation pheromone, cis-vaccenyl acetate. For EP835/Gal4, the hypermorphic mutant, all hyperexpression phenotypes were consistent with a reduced JH titer in Drosophila. Flies that hypo- or hyper- expressed P29 had a significantly shorter lifespan: Reduced lifespan correlated with increased egg production (hypomorphic flies) and hyperactivity (hypermorphic flies), respectively. Hence, the titer of P29 appeared to be positively correlated with the titer of JHE and negatively correlated with the titer of JH. Based on the collective phenotypes and detection of JHE activity in mitochondria, we hypothesize that JHE is stored in mitochondria and that P29 functions in transport of JHE to the cytosol.

1 CHAPTER 1 General introduction

Juvenile Hormone Juvenile hormone (JH) is a sesquiterpenoid insect hormone which is produced by the corpora allata in the brain. It has a methyl ester on one end and an epoxide on the other end. There are six natural forms of JH that have been detected in different orders and at different specific developmental stages: JH0, JHI, JHII, JHIII, JH bis-epoxide (JHB 3 ) and methyl farnesoate. JH I was the first form of JH to be identified (Roller and Dahm, 1968). JH has now been identified in about 100 insect species in at least 10 insect orders (Baker, 1990). JH III is the predominant homolog (Schooley et al., 1984) while other forms of JH have a more restricted distribution. All forms of JH are produced by the corpora allata. In the Diptera the corpora allata and corpora cardiaca are fused to form a ring gland. JH, in conjunction with ecdysteroids, regulates insect development, metamorphosis and reproduction. Regulation of the larval - pupal transition has been particularly well characterized. The presence of JH at a larval molt directs maintainence of the larval stage, and prevents the development of the pupal form (Kumaran, 1990). JH functions in the metamorphosis of holometabolous insects as follows: a high level of JH signals a larval/larval molt, a low level of JH signals a larval/pupal molt and no JH signals a pupal/adult molt (Schneiderman and Gilbert, 1964; Gilbert and King, 1973). In most insects, JH is involved in vitellogenesis which is required for oocyte development (Shapiro et al., 1986; Bownes and Rembold, 1987; Sappington et al., 1998). JH

2 stimulates vitellogenin production and uptake by the ovaries. JH also regulates pheromone production and calling in longer lived lepidopteran adults (McNeil, 1987; Cusson et al., 1990). However, pheromone production appears to be regulated by ecdysone in some Diptera (Barth and Lester, 1973; Jean-Marc, 1984). JH affects the behavioral response to aggregation pheromone in desert locusts (Ignell et al., 2001). JH also functions in various other processes including the development of both male and female gonadotrophic organs (Yamamoto et al., 1988), caste determination (Rachinsky et al., 1990) and diapause (Denlinger and Tanaka, 1989; Zera and Tiebel, 1989).

JH Synthesis The titer of JH is controlled by the relative rates of JH biosynthesis and degradation. JH is synthesized by the corpora allata, but is not stored there. JH biosynthesis is regulated by two kinds of peptide hormones, stimulated by allatotropins and inhibited by allatostatins, which are produced by neurosecretory cells of the brain. In the tobacco hornworm Manduca sexta, allatotropinis are produced in adults by the gene Mas-AT which encodes three prehormones which are generated by alternative splicing of the gene (Kataoka et al., 1989; Taylor et al., 1996). Allatotropic activity was found in the subesophageal ganglion of crickets (Lorenz and Hoffmann, 1995), but no gene was isolated. A 20kD peptide identified as allatotropin from Galleria mellonella larval brain, may share common epitopes with Mas-AT (Bogus and Scheller, 1996). In the vinegar fly, Drosophila melanogaster, it has been suggested that a sex-peptide synthesized by the male sex gland and transferred to the female is a source of allatotropin (Moshitsky et al., 1996). Allatostatin (Mas-AS) was purified and sequenced from M. sexta (Kramer et al.,

3 1991). In Drosophila, the Mas-AS like protein does not have an allatostatin function (Jansons et al., 1996). There is evidence for a cerebral allatostatin in D. melanogaster (Richard et al., 1990; Altaratz et al., 1991).

Proteins Involved in Juvenile Hormone Binding and Regulation Several proteins are involved in binding and metabolizing JH. Juvenile hormone binding proteins (JHBPs) identified in 1972 have been found in many species (Whitmore and Gilbert, 1972; Gilbert et al., 2000). JHBPs have high affinity for JH, but low affinity for JH degradation products. The function of JHBP is to keep JH in solution in the hemolymph, to prevent non-tissue-specific uptake of JH, to prevent non-specific degradation of JH and to assist in the interaction between JH and JH specific degradation enzymes (Goodman et al., 1990; Trowell, 1992). There are three types of hemolymph JH binding proteins. One is a 30kD protein found in Lepidoptera which has a single peptide and a single JH binding site (Kramer et al., 1974; Goodman et al., 1978). The second is lipophorin, which is a predominant hemolymph protein and main lipid carrier in the hemolymph (DeKort and Granger, 1996). The third JHBP is a 566kD protein found in Locusta migratoria that has six JH binding sites (Koopmanschap and deKort, 1988). JH degradation is attributed to juvenile hormone esterase (JHE), which is present in the hemolymph and tissues, and to juvenile hormone epoxide hydrolase (JHEH), which is tissue/membrane bound (DeKort and Granger, 1996). JHE hydrolyzes the ester of JH to produce JH acid. JHEH hydrolyzes the epoxide of JH to produce JH diol (JHD), but JHEH only functions in cells (Halarnkar et al., 1993) (Fig 1). The cumulative activities of the two enzymes convert JH to juvenile hormone acid diol (JHAD) for which no activity

4 has been discovered (Fig 2). Most JH is bound to JHBP and hence JH is protected from degradation by non-specific esterases with low binding affinities (Touhara et al., 1993; Touhara and Prestwich, 1993; Touhara et al., 1995). JHE is the only enzyme in the hemolymph that has a high affinity for JH, and hence is the only hemolymph esterase important in JH degradation (Gilbert et al., 2000) (Fig 2). A polar JH acid ester was also discovered to be synthesized and released from the corpora allata of M. sexta, but its function is unknown. (Granger et al.)

Fig 1. Primary non-oxidative metabolic pathways for JH in insects. (Halarnkar et al., 1993)

5

Fig 2. A model for JH metabolism. Bp, binding protein; EH: juvenile hormone epoxide hydrolase (Gilbert et al., 2000).

JHE Because of JHE’s important role in regulation of the JH titer in insects as well as in the development of insecticides based on JHE, this enzyme has been extensively studied in many orders of insects. JHE proteins have been isolated and purified from Trichoplusia ni (Yuhas et al., 1983; Hanzlik and Hammock, 1987; Rudnicka and Jones, 1987; Wozniak et al., 1987), Manduca sexta (Coudron et al., 1981; Venkatesh et al., 1990), Heliothesis virescens (Hanzlik et al., 1989), Leptinotarsa decemlineata (Vermunt et al., 1997), D. melanogaster (Campbell et al., 1992), Tenebrio molitor (Thomas et al., 2000) and Bombyx mori (Shiotsuki et al., 1994). Study of the house cricket (Acheta domesticus) showed that JHE activity and alpha-naphthylacetate esterase activity were regulated in the hemolymph during the first reproductive cycle. Alpha-Naphthylacetate esterases increased during the first

6 gonotrophic cycle: peaks of their activity could be observed concomitant with peaks of JHE activity (Renucci et al., 1984). JHE is present in low quantities in insect hemolymph, but since it has a high affinity for JH, even a trace amount of JH can be rapidly degraded by JHE (Ward et al., 1992). Renucci also reported the correlation between the fluctuations in JHE activity and those of hemolymph JH titers using in vitro methods (Renucci et al., 1984).

JHE Genes The JHE gene was first cloned from the tobacco budworm, H. virescens (Hanzlik et al., 1989). This moth was used because JHE in this insect has fewest isoforms (Abdel- Aal and Hammock, 1986; Hanzlik and Hammock, 1987; Abdel-Aal et al., 1988). In addition, the economic importance of this insect as an agricultural pest makes it a suitable model for the development of novel insecticides. JHE was purified and the NH 2 -terminal end was sequenced. Several DNA probes were designed based on the sequence of the protein. The strategy used to isolate a cDNA clone of JHE mRNA was to screen a cDNA expression library initially with antisera and then to re-screen the positive clones by hybridization to a mixture of the 15-mer oligonucleotides complementary to the region of the mRNA transcript coding for the NH 2 terminus of JHE protein. This increased the likelihood of obtaining a full-length clone. The positive clones were sequenced. The complete sequence of one of the clones showed a 2989-base pair insert that fit the northern blot fragments (3.0 kb) by the 15-mer oligonucleotides. The clone had an open reading frame of 1714 base pairs with a predicted mature protein of 61kDa. The translated amino acid sequence fit the sequence of the protein well. Then the H. virescens

7 JHE gene was cloned. The enzyme was similar to other carboxylesterases in the NH 2 - terminal half and in the active site, with an active site serine at position 201.

After the JHE gene had been cloned from H. virescens several other JHE genes were isolated from other insects. The Choristoneura fumiferana JHE gene was cloned by differential display of mRNAs to identify C. fumiferana genes that were induced by JH I. PCR products were then used to probe a cDNA library. The deduced amino acid sequence was similar to the H. virescens JHE sequence (Feng et al., 1999). The JHE gene of L. decemlineata was cloned by RT-PCR and RACE. The RT-PCR primer was designed based on the amino acid sequence of JHE. The product was used to screen a cDNA library to get the whole DNA and sequence (Vermunt et al., 1997). In T. ni, the partial sequence of a JHE gene has been reported (Venkataraman et al., 1994), while a cDNA clone encoding a JHE-related protein has also been reported (Jones et al., 1994). The JHE gene from M. sexta was cloned by RT-PCR using primers based on the amino acid sequence (Hinton and Hammock, 2001; Hinton and Hammock, 2003). B. mori JHE was cloned recently (Hirai et al., 2002). The H. virescens JHE cDNA was inserted into the genome of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and active JHE produced by the recombinant virus (Hammock et al., 1990).

JH in D. melanogaster The JH titer was determined for whole-body extracts at different stages of the life cycle of D. melanogaster using combined gas chromatography/selected-ion mass spectroscopy (Bownes and Rembold, 1987). Only JHIII was detected in this study. JHIII

8 was present during all larval instars but absent from eggs. The JH titer in the first and second instar larvae was higher than that in third instars. A low titer of JH was detected in prepupae but JH was undetectable in pupae. However, there was an increase in JHIII just prior to eclosion for both males and females reaching a peak just after eclosion (Fig 3). In 1989 another form of JH, JHIII bisepoxide (JHB 3 ), was isolated from the ring gland of D. melanogaster. JHB 3 may be the main biologically active form of JH in the higher Diptera. The JHIII detected previously in D. melanogaster may have resulted from instability of JHB 3 (Richard et al., 1989). JH has multiple functions in D. melanogaster. The most well known effects of JH are on pre-adult development and metamorphosis (Riddiford and Ashburner, 1991). JH has an effect on initiation and continuation of vitellogenin uptake, oocyte development and ovarian maturation (Handler and Postlethwait, 1977; Ringo et al., 2005). JH also functions in developing receptivity in females for mating, or in increasing receptivity once other factors have intiated this process (Manning, 1967; Ringo et al., 2005). The antijuvenoid Precocene I slowed ovarian growth and markedly reduced oviposition (Ringo et al., 2005).

JHE in D. melanogaster D. melanogaster JHE was purified and characterized, and is highly selective for JHIII and JHIII bisepoxide (Campbell et al., 1998). The JHE gene was identified in D. melanogaster by matching its peptide mass fingerprint with a sequence from the Drosophila genome project (Campbell et al., 2001). JHE was purified and digested with trypsin. Only one predicted gene product (CG8425) from the D. melanogaster genome

9 matched the JHE tryptic fingerprint with high confidence. A cDNA encoding this JHE was isolated using 3' and 5' RACE. This sequence is in agreement with the Drosophila genome project's prediction except that the sixth predicted intron is not removed; instead there is a stop codon followed by a polyadenylation signal and a polyA tail (Campbell et al., 2001). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) of JHE showed that the expression level of JHE in D. melanogaster is regulated by both JH and 20-hydroxyecdysone (Kethidi et al., 2005). Khlebodarova et al showed that both JHE and JHEH are related to changes in JH titer in D. melanlogaster. The high level of JH- hydrolyzing activity is determined by JHEH in adult flies (Khlebodarova et al., 1996) (Fig 4A). We determined the JHE activity in carefully staged Drosophila Oregon R prepupae and pupae. Three to five staged flies were ground in eppendorf tubes in PBS buffer on ice and centrifuged at 8000g at 4°C briefly to remove debris. The protein concentration was quantified by Bradford assay. JHE activity was measured in triplicate by a partition assay using 3 H-JH-III as substrate (Hammock and Sparks, 1977). The nanomoles of JH hydrolyzed per minute per mg protein were calculated. JHE activity reached a peak 11 hours after pupariation (Fig 4B). Our results are similar to the published data for expression of D. melanogaster JHE (Campbell et al., 1992; Khlebodarova et al., 1996). The peak JHE activity is consistent with an undetectable titer of JH in the pupal stage.

10

Fig 3. JH-III titer (- - - -) and ecdysteroid titer (-) during the life cycle of D. melanogaster (Hodgetts et al., 1977; Bownes and Rembold, 1987).

Figure 4 A. The activity of JHE and JHEH during pupal-adult development of D. melanogaster (Canton S. line). Continuous line=JHE activity, discontinuous line-JHEH activity (Khlebodarova et al., 1996).

11

Fig 4B. JHE activity assay of staged D. melanogaster Oregon R ( L: larvae; pp: prepupae; A: adult).

Nonspecific Carboxylesterases Carboxylesterases can hydrolyze carboxylate esters and play an important role in insects. Some esterases such as JHE have highly specialized functions. However, most esterases are nonspecific and can catalyze different substrates, which enable them to protect the insect against foreign substances (Satoh et al., 2002). The nonspecific carboxylesterases have broad and overlapping catalyzing activity against naturally occurring and xenobiotic esters, thioesters and amide esters (Heymann, 1982). Because of their lack of specificity, it is impossible to identify their true physiological substrates. The physiological function of the esterases may be detoxification, but there is a lack of firm evidence for this, except in the case of amplified esterases conferring resistance to organophosphorus (OP) insecticides (Long et al., 1991). Carboxylesterases have different subcellular locations: some are secreted into the blood, while others are associated with

12 the cell membrane, cytoplasm and subcellular organelles (endoplasmic reticulum). No mitochondrial esterases have been reported and no mitochondrial esterases have been identified in D. melanogaster. In a previous study, the putative JHE binding protein P29 was shown to bind JHE in M. sexta (Shanmugavelu et al., 2000). However, we found that P29 is predicted to be localized to the mitochondria. We aligned the esterases in Drosophila and found that α-E1 and cricklet which share 33% identity to JHE are predicted to be in mitochondria (MITOPROT). We hypothesize that P29 binds to α-E1 and/or cricklet in mitochondria and affects the function of α-E1 and/or cricklet.

α-esterases in D. melanogaster D. melanogaster has over 40 esterases (Oakeshott et al., 1993). Using naphthyl esters as substrates about 30 esterases have been detected by electophoretic assays (Healy et al., 1991; Spackman et al., 1994). α-esterase is so called because it can hydrolyze α- naphthyl ester. D. melanogaster has an α-esterase cluster which contains 10 active esterase genes (DmαE1 to DmαE10) and one pseudogene, dispersed over 60kb. The esterases encoded by the cluster have 37%-66% amino acid identity (Robin et al., 2000). The α-esterase cluster has about 40% of the active esterases in the genome, therefore it might be expected to play an important role in esterase function (Campbell et al., 2003). EST9 encoded by DmαE5 and EST23 encoded by DmαE7 are important esterases in Drosophila. It has been suggested they play a role in detoxification of xenobiotics or the digestion of esters (Oakeshott et al., 1993; Russell et al., 1996). This suggestion is based on the fact that EST9 and EST23 are abundant in digestive tissues and esterases are

13 important in OP insecticide resistance. α-esterase may also function in lipid metabolism because EST9 and EST23 are abundant in the fat bodies of larvae (Healy et al., 1991; Spackman et al., 1994). From screening of expressed cDNA libraries, DmαE1, DmαE2 and DmαE7 (EST23) appear to be highly expressed in the adult head, which conflicts with their role in the digestion of dietary esters (Campbell et al., 2003). Culex mosquito esterase ESTB1 has the most similar amino acid sequence to D. melanogaster αE1, which confers organophosphate resistance (Russell et al., 1996).

The Juvenile Hormone Esterase Binding Protein, P29 of M. sexta Since JHE can degrade JH and affect insect metamorphosis, a study was conducted to determine whether a recombinant baculovirus containing the JHE gene would effectively kill Lepidoptera, based on the hypothesis that increased JHE titers would reduce JH titers at an inappropriate time and be toxic to insects. Baculoviruses infect insects primarily within the Lepidoptera. However, the recombinant baculovirus that expressed wild-type JHE had no noticeable effect on the survival time of infected larvae relative to the wild type, possibly because JHE is rapidly removed from the hemolymph. Three modified JHEs were produced with conservative changes to avoid disruption of the three-dimensional structure or the catalytic activity of JHE. Infection of larvae with a baculovirus expressing one of the mutated JHEs (JHE-KK) decreased feeding damage by 50% (Bonning et al., 1997). JHE-KK had both Lys 29 and Lys 524

replaced with arginines. Analysis of pericardial cells exposed to either the wild-type JHE or the mutated form of JHE by electron microscopy showed that the mutation caused failure of the normal lysosomal targeting of JHE in pericardial cells (Bonning et al.,

14 1997). JHE-KK was scattered in pericardial cells, and accumulation of JHE-KK in the lysosomes was five-fold less than for wild type JHE. Uptake of JHE-KK from the hemolymph was not affected by the mutations made. Pericardial cells take up JHE by endocytosis, which suggested that JHE was specifically binding to pericardial cells by receptors (Ichinose et al., 1993; Mellman, 1996; Bonning et al., 1997). To investigate possible binding proteins of JHE, a recombinant cDNA phage display library of M. sexta pericardial cells was screened and a sequence encoding a 29 kDa binding protein identified (GenBank database accession number AF153450) (Shanmugavelu et al., 2000). P29 was confirmed to be present in the pericardial cells and fat body tissue of M. sexta larvae by western and northern blots. Interaction of P29 with recombinant H. virescens-derived JHE was confirmed in vivo and in vitro by immunoprecipitation with antisera that recognized both H. virescens JHE and M. sexta P29. Based on the previous result that JHE-KK had reduced targeting to lysosomes compared to wild type JHE and was insecticidal, the binding of P29 to JHE and JHE-KK was tested. P29 bound less effectively to JHE-KK than to wild type JHE (Shanmugavelu et al., 2001). With this much data pointing to P29 as a significant potential intermediate in the degradation process of JHE, information about P29’s pathway and the location of P29’s interaction with JHE may provide a better understanding of how the titer of JHE can be manipulated.

Misexpression of proteins in Drosophila A successful system for misexpression of specific proteins in Drosophila was introduced by Rørth (1996). A transposable element (EP element), which contains UAS

15 sites that can bind to the yeast transcription factor Gal4 and a promoter, was randomly inserted into the 5’ untranslated region of genes. The insertion would affect expression of the EP tagged gene (the gene immediately downstream of the EP element). Furthermore, the EP element would allow for the gene immediately downstream to be overexpressed and misexpressed by crossing to lines with gal4-expressing insertions. Temporal control

of gene expression in Drosophila has generally been accomplished

by using a heat shock (hs) promoter; spatial control of gene expression has been accomplished by using tissue- specific promoters (Joseph, 2002).

D. melanogaster P29 Homolog Following the release of the D. melanogaster genome sequence (Adams et al., 2000), a P29 homolog was identified that allowed us to exploit the knowledge of Drosophila genetics and associated research tools to determine the role of P29. The gene product of CG3776 (Dm) shares 49% identity with M. sexta P29 (Ms) over 206 residues. MsP29 has 243 amino acids while CG3776 encodes a protein with 263 amino acids resulting in a calculated molecular mass of 30kD. The D. melanogaster P29 gene CG3776 is located on the right arm of the second chromosome at position 60E10. This region is a gene-dense region. At the 5’ end of CG3776 is the RpL19 gene which encodes a ribosome protein, L19. At the 3’ end of CG3776 is the Phk-3 gene encoding a protein pherokine-3, which is a putative odor/pheromone binding protein (Sabatier et al., 2003) (Fig 5). There are two D. melanogaster EP lines (EP835 and EP840) available at the Szeged Drosophila Stock Centre. Both of the EP lines are homozygous viable. EP835 has an EP element inserted

16 into the chromosome 35nt upstream of the CG3776 start site in the 5’ untranslated region (Fig 5). The EP element of EP840 is inserted into the chromosome 2nt upstream from that of EP835 (i.e. 37 nt upstream of the start site). EP835 was used to make a double mutant with the pnr gene, which encodes a zinc-finger protein with homology to the vertebrate GATA transcription factors (Ramain et al., 1993; Winick et al., 1993). Hyperexpression of EP835 suppressed the pnrGal4/+ phenotype, suggesting that P29 interacts with this transcription factor (Pena-Rangel et al., 2002). However, no gene function studies have been conducted for CG3776 using this EP line. We used these lines to study gene function when hypoexpressed. It is estimated that more than two thirds of genes in Drosophila have no obvious loss of function phenotype, possibly due to functional redundancy (Miklos and Rubin, 1996). In such cases, overexpression of a gene can provide an indication of function (Rorth, 1996) (Fig 6). In addition, the EP line can be used to generate ‘imprecise excisions’ by remobilizing the P element, which may result in a complete null mutant.

Fig 5. Diagram of CG3776 and its neighboring genes (From chromosome 2R: 20,476k to 20479.5k; adapted from flybase). The EP element insertion site of EP835 is indicated.

17

Fig 6. Outline of the overexpression screen. The EP line contains a single EP element inserted into the P29 5’ untranslated region. When mated with GAL4 flies, progeny with straight wings will contain both the Gal4 and EP elements. This allows GAL4 to bind to GAL4 binding sites within the EP element, thereby inducing the EP promoter to transcribe the gene immediately adjacent to the element (P29).

Structural Analysis of Drosophila and Manduca P29 MITOP (mitochondria project) is a database for mitochondria-related proteins in selected species. MITOPROT is a program that can predict mitochondrial targeting sequences by analyzing the N-terminal sequence (Claros and Vincens, 1996) (http://ihg.gsf.de/ihg/mitoprot.html). Mitochondrial targeting peptides are known to be rich in arginine, alanine, and serine, while negatively charged amino acid residues (aspartic acid and glutamic acid) are rare (von Heijne et al., 1989). Further, these residues are believed to form an amphiphilic α-helix important for import into mitochondria (Nakai and Kanehisa, 1992; Bannai et al., 2002). The Drosophila P29 protein (DmP29) is predicted with 93% probability to be a mitochondrial protein and Manduca P29 (MsP29) has 99.6% probability to be a mitochondrial protein (Table 1). The predicted mitochondrial signal sequence of DmP29 is 54 residues. The signal peptide is therefore

18 ~5.8kD. Therefore, the mature DmP29 should have a calculated molecular mass of 24.2kD. This result made us review the former experiments in M. sexta (Shanmugavelu et al., 2000). JHE and MsP29 can interact in vitro as detected by in vitro immunoprecipitation. In the in vivo immunoprecipitation experiment, biotinylated JHE was injected into Manduca larvae. Tissues were homogenized and precipitated with P29 antibody. If P29 is located in mitochondria it would be released by homogenization and then able to interact with JHE. Hence the data can be explained even if JHE and P29 are in different subcellular locations and do not bind in vivo. Table 1. Predicted mitochondrial signal sequences of D. melanogaster and M. sexta P29 (MITOPROT) Insect Probability of export to mitochondria Cleavage site Predicted mitochondrial signal sequence

D. melanogaster 93.0% 55 MQHTLIRCLGMARISLMRLQPRP TVAASGGQEAGSISKPTQPVSR SFASLPQEQ M. sexta 99.6% 44 MNLALRQVLTRQSFRLCDRYA HKNVAKQIPLTSQCSVIQYRKY

Full document contains 169 pages
Abstract: Insect development, metamorphosis and reproduction are regulated in part by the action of juvenile hormone (JH). The titer of JH is regulated in turn by the action of the enzymes juvenile hormone epoxide hydrolase and juvenile hormone esterase (JHE). Because of the potential for disruption of regulation of insect development through perturbation of the action of JH, the biology of JHE has been well studied. A putative juvenile hormone esterase binding protein, P29 was identified in the tobacco hornworm, Manduca sexta. Following sequencing of the Drosophila melanogaster genome, we identified a homolog of P29 in D. melanogaster, and used this insect for analysis of the biology and function of P29 in relation to JHE. The gene encoding D. melanogaster P29 (DmP29), CG3776 was cloned, recombinant DmP29 expressed in E. 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 30kD. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6kD N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25kD protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five(TM) 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. We investigated the potential ligands of DmP29 by testing three hypotheses: (i) DmP29 binds to D. melanogaster JHE: We produced a stably transformed insect cell line that expresses DmJHE and confirmed that DmP29 binds to D. melanogaster P29. DmJHE binds to both the 25 kD and 50 kD immunoreactive proteins. (ii) DmP29 binds other, non-specific esterases including two esterases predicted to be targeted to the mitochondria: We did not detect any interaction between DmP29 and non-specific esterases. (iii) DmP29 binds to other proteins in D. melanogaster : Ligand blot analysis, immunoprecipitation experiments and affinity binding experiments showed that larval serum protein 1 binds the 25 kD P29. The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolysed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant, given the high abundance, and "sticky" nature of these proteins. Interaction of DmP29 with LSP may result from non-specific associations. We used P29 hypo- and hyper-expression mutants to elucidate the function of P29 and the potential interaction of P29 with JHE. The hypomorphic mutant EP835 of P29 had reduced JHE activity when compared to wild type flies. Hyperexpression of P29 in EP/Gal4 during the early larval stages was lethal, while hyperexpression during the third instar resulted in reduced size of adult flies. This phenotype showed that overexpression of P29 interfered with insect development. Hyperexpression in newly eclosed but not in older females resulted in reduced fecundity, indicating that overexpression of P29 affected ovarian development. Fecundity was not affected by P29 hyperexpression in the male. Hypermorphic adults exhibited male-male courtship behavior. Hyperexpressed females showed reduced receptivity to males. Hyperexpressed females had decreased production of courtship pheromone, cis, cis-7, 11-hepta cosadiene, which resulted in male flies being unable to locate female flies. Hyperexpression of P29 in males resulted in decreased production of the aggregation pheromone, cis-vaccenyl acetate. For EP835/Gal4, the hypermorphic mutant, all hyperexpression phenotypes were consistent with a reduced JH titer in Drosophila. Flies that hypo- or hyper-expressed P29 had a significantly shorter lifespan: Reduced lifespan correlated with increased egg production (hypomorphic flies) and hyperactivity (hypermorphic flies), respectively. Hence, the titer of P29 appeared to be positively correlated with the titer of JHE and negatively correlated with the titer of JH. Based on the collective phenotypes and detection of JHE activity in mitochondria, we hypothesize that JHE is stored in mitochondria and that P29 functions in transport of JHE to the cytosol.