Functional Characterization of the Bombyx mori Fatty Acid Transport Protein (BmFATP) within the Silkmoth Pheromone Gland
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m806072200
ISSN1083-351X
AutoresAtsushi Ohnishi, Kana Hashimoto, Kiyohiro Imai, Shogo Matsumoto,
Tópico(s)Insect-Plant Interactions and Control
ResumoFatty acid transport protein (FATP) is an evolutionarily conserved membrane-bound protein that facilitates the uptake of extracellular long chain fatty acids. In humans and mice, six FATP isoforms have been identified and their tissue-specific distributions suggest that each plays a discrete role in lipid metabolism in association with fatty acid uptake. While the presence of FATP homologs in insects has been demonstrated, their functional role remains to be characterized. Pheromonogenesis is defined as the dynamic period in which all machinery required for sex pheromone biosynthesis is generated and organized within the pheromone gland (PG) cells. By exploiting this unique system in the PG of the silkmoth, Bombyx mori, we found that BmFATP is predominantly expressed in the PG and undergoes up-regulation 1 day prior to eclosion. Before eclosion, B. mori PG cells accumulate cytoplasmic lipid droplets (LDs), which play a role in storing the pheromone (bombykol) precursor fatty acid in the form of triacylglycerol. RNAi-mediated gene silencing of BmFATP in vivo significantly suppressed LD accumulation by preventing the synthesis of triacylglycerols and resulted in a significant reduction in bombykol production. These results, in conjunction with the findings that BmFATP stimulates the uptake of extracellular long-chain fatty acids and BmFATP knockdown reduces cellular long-chain acyl-CoA synthetase activity, suggest that BmFATP plays an essential role in bombykol biosynthesis by stimulating both LD accumulation and triacylglycerol synthesis via a process called vectorial acylation that couples the uptake of extracellular fatty acids with activation to CoA thioesters during pheromonogenesis. Fatty acid transport protein (FATP) is an evolutionarily conserved membrane-bound protein that facilitates the uptake of extracellular long chain fatty acids. In humans and mice, six FATP isoforms have been identified and their tissue-specific distributions suggest that each plays a discrete role in lipid metabolism in association with fatty acid uptake. While the presence of FATP homologs in insects has been demonstrated, their functional role remains to be characterized. Pheromonogenesis is defined as the dynamic period in which all machinery required for sex pheromone biosynthesis is generated and organized within the pheromone gland (PG) cells. By exploiting this unique system in the PG of the silkmoth, Bombyx mori, we found that BmFATP is predominantly expressed in the PG and undergoes up-regulation 1 day prior to eclosion. Before eclosion, B. mori PG cells accumulate cytoplasmic lipid droplets (LDs), which play a role in storing the pheromone (bombykol) precursor fatty acid in the form of triacylglycerol. RNAi-mediated gene silencing of BmFATP in vivo significantly suppressed LD accumulation by preventing the synthesis of triacylglycerols and resulted in a significant reduction in bombykol production. These results, in conjunction with the findings that BmFATP stimulates the uptake of extracellular long-chain fatty acids and BmFATP knockdown reduces cellular long-chain acyl-CoA synthetase activity, suggest that BmFATP plays an essential role in bombykol biosynthesis by stimulating both LD accumulation and triacylglycerol synthesis via a process called vectorial acylation that couples the uptake of extracellular fatty acids with activation to CoA thioesters during pheromonogenesis. Lepidopteran sex pheromones are often used by females to lure conspecific males, with most sharing a common progenitor that is de novo synthesized from acetyl-CoA via fatty acid synthesis in the pheromone gland (PG), 2The abbreviations used are: PG, pheromone gland; ACBP, acyl-CoA-binding protein; ACS, acyl-CoA synthetase; Bmpgdesat1, B. mori pheromone gland desaturase 1; DEPC, diethyl pyrocarbonate; ELSD, evaporative light scattering detector; FATP, fatty acid transport protein; LCFA, long-chain fatty acid; LD, lipid droplet; mgACBP, midgut ACBP; MS, mass spectrometry; PBAN, pheromone biosynthesis activating neuropeptide; PBANR, PBAN receptor; pgACBP, PG-specific ACBP; pgFAR, PG-specific fatty acyl reductase; RNAi, RNA interference; TG, triacylglycerol; VLACS, very-long-chain acyl-CoA synthetase; RACE, rapid amplification of cDNA ends; PBS, phosphate-buffered saline; UTR, untranslated region. a functionally differentiated organ located in the proximity of the terminal abdominal segment. To date, sex pheromones from more than 570 moth species have been chemically identified with most species utilizing Type I pheromone components, which consist of straight-chain compounds 10–18 carbons in length with an oxygenated functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds (1Jurenka R.A. Insect Pheromone Biochemistry and Molecular Biology. Elsevier Academic Press, Oxford, UK2003: 53-80Crossref Scopus (81) Google Scholar). Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multicomponent blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends (1Jurenka R.A. Insect Pheromone Biochemistry and Molecular Biology. Elsevier Academic Press, Oxford, UK2003: 53-80Crossref Scopus (81) Google Scholar, 2Rafaeli A. Int. Rev. Cytol. 2002; 213: 49-91Crossref PubMed Scopus (93) Google Scholar, 3Tillman J.A. Seybold S.J. Jurenka R.A. Blomquist G.J. Insect. Biochem. Mol. Biol. 1999; 29: 481-514Crossref PubMed Scopus (436) Google Scholar). However, the molecular mechanisms underlying sex pheromone production in moth PG cells have remained poorly understood. The PG of the silkmoth, Bombyx mori, originates from the intersegmental membrane between the eighth and ninth abdominal segments and can be characterized as a pair of eversible, ventrolateral sacs (sacculi laterales) consisting of about 10,000 single-layered epidermal cells that are in direct contact with the overlying cuticular surface (4Fónagy A. Yokoyama N. Okano K. Tatsuki S. Maeda S. Matsumoto S. J. Insect. Physiol. 2000; 46: 735-744Crossref PubMed Scopus (45) Google Scholar). These cells produce and release the principle sex pheromone component bombykol, (E,Z)-10,12-hexadecadien-1-ol, which is synthesized de novo via acetyl-CoA derived palmitate (16:Acyl) by the sequential actions of a bifunctional Δ10,12-desaturase and a fatty-acyl reductase, both of which have been recently characterized as Bmpgdesat1 and PG-specific fatty acyl reductase (pgFAR), respectively (5Ando T. Hase T. Funayoshi A. Arima R. Uchiyama M. Agric. Biol. Chem. 1988; 52: 141-147Google Scholar, 6Moto K. Suzuki M.G. Hull J.J. Kurata R. Takahashi S. Yamamoto M. Okano K. Imai K. Ando T. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8631-8636Crossref PubMed Scopus (99) Google Scholar, 7Moto K. Yoshiga T. Yamamoto M. Takahashi S. Okano K. Ando T. Nakata T. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9156-9161Crossref PubMed Scopus (145) Google Scholar). As with most lepidopteran sex pheromone biosyntheses (2Rafaeli A. Int. Rev. Cytol. 2002; 213: 49-91Crossref PubMed Scopus (93) Google Scholar, 8Rafaeli A. Jurenka R.A. Insect Pheromone Biochemistry and Molecular Biology. Elsevier Academic Press, Oxford, UK2003: 107-136Crossref Scopus (91) Google Scholar), bombykol biosynthesis is triggered by a molecular interaction between the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN), and its cognate PG cell-surface receptor, the PBAN receptor (PBANR), which has also recently been characterized as a G protein-coupled receptor (GPCR) that belongs to the neuromedin U receptor family (9Choi M.Y. Fuerst E.J. Rafaeli A. Jurenka R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9721-9726Crossref PubMed Scopus (141) Google Scholar, 10Hull J.J. Ohnishi A. Moto K. Kawasaki Y. Kurata R. Suzuki M.G. Matsumoto S. J. Biol. Chem. 2004; 279: 51500-51507Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Kim Y.-J. Nachman R.J. Aimanova K. Gill S. Adams M.E. Peptides. 2008; 29: 268-275Crossref PubMed Scopus (65) Google Scholar). Pheromonogenesis can be defined as the dynamic period in which all machinery required for sex pheromone biosynthesis is generated and organized within the PG cells of the female moth before and after eclosion (12Matumoto S. Hull J.J. Ohnishi A. Moto K. Fónagy A. J. Insect. Physiol. 2007; 53: 752-759Crossref PubMed Scopus (49) Google Scholar). Before eclosion, bombykol-producing cells can be characterized by the abundance of lipid droplets (LDs) within the cytoplasm; these LDs begin to form one or 2 days prior to eclosion and they rapidly accumulate on the day of eclosion. In contrast, the density of the LDs decreases in response to PBAN stimulation after eclosion (4Fónagy A. Yokoyama N. Okano K. Tatsuki S. Maeda S. Matsumoto S. J. Insect. Physiol. 2000; 46: 735-744Crossref PubMed Scopus (45) Google Scholar, 13Fónagy A. Yokoyama N. Matsumoto S. Arthropod. Struct. Dev. 2001; 30: 113-123Crossref PubMed Scopus (39) Google Scholar). We have analyzed the chemical composition of the LDs and confirmed that they are composed of various triacylglycerols (TGs) with the bombykol precursor, Δ10, 12-hexadecadienoate, predominantly sequestered as a major component at the sn-1 and/or sn-3 position of the glycerides, indicating that the LDs play an essential role in storing the bombykol precursor in the form of TGs and releasing it for bombykol production in response to PBAN stimulation (14Matsumoto S. Fónagy A. Yamamoto M. Wang F. Yokoyama N. Esumi Y. Suzuki Y. Insect. Biochem. Mol. Biol. 2002; 32: 1447-1455Crossref PubMed Scopus (41) Google Scholar). In addition, we have recently established a method for targeted disruption of B. mori PG-specific genes in vivo by the use of RNA interference (RNAi) and have successfully demonstrated that loss of function of the PG-specific acyl-CoA-binding protein (pgACBP) prevents LD accumulation, and consequently, limits the availability of the bombykol precursors (15Ohnishi A. Hull J.J. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4398-4403Crossref PubMed Scopus (107) Google Scholar). However, the precise mechanisms underlying LD accumulation in the PG cells prior to eclosion have yet to be characterized. Fatty acid transport proteins (FATPs) belong to an evolutionarily conserved family of membrane-bound proteins that facilitate the uptake of extracellular long-chain fatty acids (LCFAs) and/or very LCFAs and catalyze the ATP-dependent esterification of these fatty acids to their corresponding acyl-CoA derivatives (16Hall A.M. Smith A.J. Bernlohr D.A. J. Biol. Chem. 2003; 278: 43008-43013Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar). In humans and rodents, six related members of FATPs (FATP1 to FATP6) have been identified and their tissue expression patterns have been analyzed (18Stahl A. Pflugers Arch. 2004; 447: 722-727Crossref PubMed Scopus (237) Google Scholar). Although FATP homologs are also present in several insect species (17Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar, 19Doege H. Stahl A. Physiology. 2006; 21: 259-268Crossref PubMed Scopus (173) Google Scholar), their functional role has yet to be characterized. To identify and characterize functional proteins involved in the pheromonogenesis of the silkmoth, B. mori, we have generated a PG expressed-sequence tag (EST) data base by constructing a normalized PG cDNA library prepared from newly emerged female moths of the inbred B. mori strain (p50) (20Yoshiga T. Okano K. Mita K. Shimada T. Matsumoto S. Gene. 2000; 246: 339-345Crossref PubMed Scopus (50) Google Scholar). In the course of expression analyses of these EST clones using various tissues as well as PGs isolated during different developmental stages, we found that a gene encoding a FATP homolog in B. mori (BmFATP) is predominantly expressed in the PG. In this study, we sought to examine the functional role of BmFATP in B. mori pheromonogenesis. Insects—Larvae of the inbred p50 strain of B. mori, kindly provided by T. Shimada of the University of Tokyo, were reared on mulberry leaves and maintained under a 16L:8D photoperiod at 25 °C. Pupal age was distinguished based on morphological characteristics as described (14Matsumoto S. Fónagy A. Yamamoto M. Wang F. Yokoyama N. Esumi Y. Suzuki Y. Insect. Biochem. Mol. Biol. 2002; 32: 1447-1455Crossref PubMed Scopus (41) Google Scholar). Rapid Amplifying cDNA Ends (RACE)—Messenger RNA was isolated from about 100 μg of PG total RNA by using a Micro-Fast Track kit (Invitrogen, Carlsbad, CA). RACE was performed using a GeneRacer kit (Invitrogen) according to the manufacturer's instructions. Computer-assisted sequence analyses were performed using GENETYX-MAC Ver.12.0 (Software Development Co., Tokyo, Japan). RT-PCR Analysis—PGs and other tissues were dissected into insect Ringer's solution (35 mm NaCl, 36 mm KCl, 12 mm CaCl2, 16 mm MgCl2, 274 mm glucose, and 5 mm Tris-HCl, pH 7.5) and mechanically trimmed as described (21Ozawa R. Matsumoto S. Insect. Biochem. Mol. Biol. 1996; 26: 259-265Crossref PubMed Scopus (36) Google Scholar). Total RNA was isolated from the trimmed PGs by the method of Chomczynski and Sacchi (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) and first-strand cDNA synthesis was performed using an RNA PCR kit (Takara Bio Inc.) according to the manufacturer's instructions with 500 ng of total RNA. Fragments of BmFATP were amplified from oligonucleotide primer pairs designed from published sequences (NRPG1860-F1 [sense primer]: 5′-TATACACGTCAGGGACTACTG-3′ and NRPG1860-R1 [antisense primer]: 5′-TTCAACTCCTAAGGGCACGT-3′). PCR was performed under the conditions of 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s. After PCR, 5 μl of the PCR products were electrophoresed on a 1.5% agarose gel in TAE buffer. Synthesis and Injection of dsRNA—The templates for synthesis of dsRNAs corresponding to BmFATP were prepared using gene-specific primers containing T7 polymerase sites (NRPG1860T7-F1 [sense primer]: 5′-CCTAATACGACTCACTATAGGGCGGTATACACGTCAGGGA-3′ and NRPG1860T7-R1 [antisense primer]: 5′-CCTAATACGACTCACTATAGGGCGGTTCAACTCCTAAGGG-3′; nucleotide sequences corresponding to the T7 promoter region are underlined). PCR was performed under the conditions of 6 cycles of 94 °C for 30 s, 56.5 °C for 30 s, 68 °C for 90 s followed by 30 cycles of 94 °C for 30 s, 66 °C for 30 s, 68 °C for 90 s using KOD-Plus-(Toyobo, Osaka, Japan) with the resulting products purified (Wizard SV Gel and PCR Clean-Up kit, Promega, Madison, WI) and used as templates to generate dsRNAs using the AmpliScribe™ T7 High Yield Transcription kit (Epicenter Technologies, Madison, WI) according to the manufacturer's instructions. After synthesis, the dsRNAs were diluted with diethyl pyrocarbonate-(DEPC-)treated H2O, the RNA concentrations measured (A260), and the products were analyzed by gel electrophoresis to confirm annealing. Samples were diluted to the desired concentration (final volume 2 μl) and injected near the abdominal tip of 1-day-old pupae (i.e. pupae 1 day removed from the larval-pupal molt) using a 10-μl microsyringe (Hamilton). Control pupae were injected with 2 μl of DEPC-treated H2O alone. After injection, pupae were maintained under normal conditions until adult emergence. In Vivo Bombykol Analysis—Adult females were decapitated within 3 h of emergence and maintained at 25 °C for 24 h. They were then injected with either 5 pmol (2 μl) B. mori PBAN in PBS or PBS alone. Abdominal tips were dissected 90 min after injection and bombykol production was measured by HPLC as described (23Matsumoto S. Kitamura A. Nagasawa H. Kataoka H. Orikasa C. Mitsui T. Suzuki A. J. Insect. Physiol. 1990; 36: 427-432Crossref Scopus (186) Google Scholar) using a Senshu-Pac NO2 column (Senshu Scientific Co., Tokyo, Japan). Microscopic Examination of Cytoplasmic Lipid Droplets—Abdominal tips were dissected and mechanically trimmed from normal, decapitated, or RNAi-treated females. The excised glands were fixed with a 4% formalin/PBS solution and stained with Nile Red (a fluorescent probe for intracellular neutral lipids; Molecular Probes Inc. Eugene, OR) as described (13Fónagy A. Yokoyama N. Matsumoto S. Arthropod. Struct. Dev. 2001; 30: 113-123Crossref PubMed Scopus (39) Google Scholar). Fluorescence microscopy was performed with an OLYMPUS BX-60 system equipped with a PM-30 exposure unit and a BH20-RFL-T3 light source (400× magnification). Nile Red imaging was performed with a 330–385-nm band pass excitation filter, a 400-nm dichroic mirror, and a 420-nm long pass barrier filter (OLYMPUS cube WU). Images were processed and merged using Photoshop CS (Adobe Systems Inc., San Jose, CA). Analysis of Lipid Droplet Components—Five trimmed PGs were prepared from the desired stages of female moths and dipped in 100 μl of acetone for 10 min at room temperature. The dried acetone extracts were dissolved in n-hexane and loaded on either a Senshu-Pak PEGASIL-Silica 120–5 column (Senshu Sci. Co.; 4.6 mm i.d. × 250 mm, pore size: 120 Å) equilibrated with n-hexane/acetic acid (99/1) or a SSC-C22 docosil column (Senshu Sci. Co.; 4.6 mm i.d. × 250 mm, pore size: 120 Å) equilibrated with acetonitrile/ethanol (6/4). TG components were separated as described previously (14Matsumoto S. Fónagy A. Yamamoto M. Wang F. Yokoyama N. Esumi Y. Suzuki Y. Insect. Biochem. Mol. Biol. 2002; 32: 1447-1455Crossref PubMed Scopus (41) Google Scholar) and detected using an evaporative light scattering detector (ELSD; SEDEX model 75, Sedere, France). Fatty acyl groups in the TGs were identified with Fast Atom Bombardment mass spectrometry (MS) and tandem MS (MS-MS) analyses using a JEOL (Tokyo, Japan) JMS HX/HX-110A tandem mass spectrometer as described (14Matsumoto S. Fónagy A. Yamamoto M. Wang F. Yokoyama N. Esumi Y. Suzuki Y. Insect. Biochem. Mol. Biol. 2002; 32: 1447-1455Crossref PubMed Scopus (41) Google Scholar). Analysis of Cellular Fatty Acid Uptake using the Fluorescent Fatty Acid BODIPY 500/510 C1,C12—Trimmed PGs were prepared from normal or RNAi-treated females 12 h after eclosion and maintained at room temperature for 2 h in insect Ringer's solution. The PGs were then incubated with 5 μm BODIPY 500/510 C1,C12 (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-sindacene-3-dodecanoic acid, Molecular Probes, D3823) in 50 μl of insect Ringer's solution for 10 min in the dark. After incubation, the PGs were washed twice with PBS containing 15 μm fatty acid-free bovine serum albumin and then with PBS alone (24Zou Z. DiRusso C.C. Ctrnacta V. Black P.N. J. Biol. Chem. 2002; 277: 31062-31071Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). All steps were performed at room temperature. Incorporation of BODIPY 500/510 C1,C12 was visualized on an OLYMPUS BX-60 system equipped with a PM-30 exposure unit and a BH20-RFL-T3 light source (400× magnification). Fluorescent imaging was performed with a 330–385-nm band pass excitation filter, a 400-nm dichroic mirror, and a 420-nm long pass barrier filter. Images were processed and merged using Photoshop CS. Measurement of Radiolabeled Fatty Acid Uptake—Rates of fatty acid transport were determined essentially as described by DiRusso et al. (25DiRusso C.C. Connell E.J. Faergeman N.J. Knudsen J. Hansen J.K. Black P.N. Eur. J. Biochem. 2000; 267: 4422-4433Crossref PubMed Scopus (42) Google Scholar). [9,10-3H]Palmitic acid (47.7 Ci/mmol) and [9,10-3H]oleic acid (45.5 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Each trimmed PG, prepared from either a normal or RNAi-treated female moth 12 h after eclosion, was preincubated for 2 h at room temperature in 250 μl of insect Ringer's solution containing 15 μm fatty acid-free bovine serum albumin, and the assay was initiated by the addition of 80 nm [3H]palmitic acid or [3H]oleic acid. After incubation, each PG was washed three times with PBS containing 0.5% Brji 58, lysed with 200 μl 0.3 n NaOH, and the cell-associated radioactivity was measured by liquid scintillation counting. Measurement of Fatty Acyl-CoA Synthetase (ACS) Activity—Fatty ACS activity was assayed based on the conversion of [3H]palmitic acid or [3H]oleic acid to their CoA derivatives using a modified method from Hall et al. (16Hall A.M. Smith A.J. Bernlohr D.A. J. Biol. Chem. 2003; 278: 43008-43013Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Each PG prepared from normal or RNAi-treated females 12 h after eclosion was homogenized in 25 μl of 30 mm NaCl, 1% Triton X-100, and 100 mm Tris-HCl, pH 7.5. The homogenate was then incubated at 30 °C for 5 min with 250 μl of 30 mm NaCl, 100 mm Tris-HCl, pH 7.5, 80 nm 3H-labeled fatty acid, 10 mm ATP, 5 mm MgCl2, 200 μm CoA, and 200 μm dithiothreitol. The reaction was terminated with the addition of 1.25 ml of isopropanol:heptane: H2SO4 (40:10:1, v/v/v), 0.5 ml H2O, and 0.75 ml of heptane to facilitate organic phase separation. The aqueous phase was extracted three times with 0.75 ml of heptane to remove unreacted fatty acids, and the radioactivity was determined by liquid phase scintillation counting. Cloning of the Bombyx mori FATP Gene (BmFATP) Expressed in the PG—We found a cDNA clone expressed in the PG that encodes the FATP homolog in B. mori, which we have designated as BmFATP. This clone (NRPG1860) is present in the B. mori PG EST data base, which was constructed from our normalized p50 PG cDNA library (20Yoshiga T. Okano K. Mita K. Shimada T. Matsumoto S. Gene. 2000; 246: 339-345Crossref PubMed Scopus (50) Google Scholar) and then integrated into the public B. mori EST data base (SilkBase). Subsequent RT-PCR amplification and both 3′- and 5′-RACE of NRPG1860 indicated that BmFATP is comprised of a 2,094-nt open reading frame encoding a 698 amino acid protein flanked by a 233-nt 5′-UTR and a 851-nt 3′-UTR. Comparison of the BmFATP sequence with sequences from other organisms revealed high similarities with the Apis mellifera FATP, Anopheles gambiae FATP, Drosophila melanogaster FATP, Mus musculus FATP4, and Saccharomyces cerevisiae Fat1p (55, 54, 53, 42, and 29% similarity, respectively) (Fig. 1). These FATPs, including BmFATP, can be characterized by two highly conserved sequences, the ATP/AMP signature motif common to the adenylate-forming enzymes and the FATP/VLACS signature motif (24Zou Z. DiRusso C.C. Ctrnacta V. Black P.N. J. Biol. Chem. 2002; 277: 31062-31071Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) (Fig. 1). Two studies have described the topology of the FATPs, one for murine FATP1 (26Lewis S.E. Listenberger L.L. Ory D.S. Schaffer J.E. J. Biol. Chem. 2001; 276: 37042-37050Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and the second for yeast Fat1p (27Obermeyer T. Fraisl P. DiRusso C.C. Black P.N. J. Lipid Res. 2007; 48: 2354-2364Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Analysis of BmFATP with the membrane protein structure prediction program SOSUI revealed that BmFATP contains two possible N-terminal transmembrane domains (amino acid residues 42–64 and 68–90) (data not shown), suggesting that the BmFATP more closely resembles that found in yeast. The yeast Fat1p has two transmembrane-spanning domains, and experimental data have suggested that both N- and C-terminal regions are positioned on the inner face of the plasma membrane, with the ATP/AMP and FATP/VLACS domains of Fat1p facing the cytoplasm (27Obermeyer T. Fraisl P. DiRusso C.C. Black P.N. J. Lipid Res. 2007; 48: 2354-2364Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A search for FATP gene homologs in the public B. mori genome data base (Silkworm Genome Database) suggested that the BmFATP is a single-copy gene. Expression Analyses of BmFATP using RT-PCR—To examine the tissue distribution of the BmFATP transcript, we performed RT-PCR analyses using various tissues prepared from p50 female adults and larvae (Fig. 2). In adult tissues prepared 3 h after eclosion, we found that the BmFATP transcript was predominantly expressed in the PG with a less intense signal detectable in other tissues including midgut and fat body (Fig. 2A). Stage-specific expression of BmFATP within the PG indicated that it undergoes significant up-regulation 1 day prior to adult emergence (Fig. 2B). This up-regulation in the PG is reminiscent of PG-specific proteins crucial to B. mori pheromonogenesis, i.e. pgFAR, Bmpgdesat1, pgACBP, mgACBP, and PBANR (6Moto K. Suzuki M.G. Hull J.J. Kurata R. Takahashi S. Yamamoto M. Okano K. Imai K. Ando T. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8631-8636Crossref PubMed Scopus (99) Google Scholar, 7Moto K. Yoshiga T. Yamamoto M. Takahashi S. Okano K. Ando T. Nakata T. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9156-9161Crossref PubMed Scopus (145) Google Scholar, 10Hull J.J. Ohnishi A. Moto K. Kawasaki Y. Kurata R. Suzuki M.G. Matsumoto S. J. Biol. Chem. 2004; 279: 51500-51507Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 28Matsumoto S. Yoshiga T. Yokoyama N. Iwanaga M. Koshiba S. Kigawa T. Hirota H. Yokoyama S. Okano K. Mita K. Shimada T. Tatsuki S. Insect. Biochem. Mol. Biol. 2001; 31: 603-609Crossref PubMed Scopus (49) Google Scholar). Accordingly, these results suggest the functional involvement of BmFATP in B. mori pheromonogenesis as well. Apart from expression in the PG, we also found the BmFATP transcript prominently expressed in the larval midgut and fat body (Fig. 2C). In addition, a weak but steady signal was detected in the larval brain and silk gland (Fig. 2C). RNAi-mediated Knockdown of BmFATP Transcripts in Vivo: Effect on Bombykol Production—We have recently established a method for RNAi-mediated knockdown of B. mori PG-specific genes in vivo (15Ohnishi A. Hull J.J. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4398-4403Crossref PubMed Scopus (107) Google Scholar). To elucidate the in vivo function of the BmFATP in the PG during pheromonogenesis, we synthesized double-stranded RNA (dsRNA) corresponding to the open reading frame of BmFATP and examined its inhibitory effect on bombykol production in vivo by using our RNAi protocol (15Ohnishi A. Hull J.J. Matsumoto S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4398-4403Crossref PubMed Scopus (107) Google Scholar). When we injected 10 μg of dsRNA for BmFATP into 1-day-old p50 female pupae (i.e. pupae 1 day removed from the larval-pupal molt), BmFATP mRNA levels in the PG after eclosion were prominently reduced compared with the control pupae injected with DEPC-treated H2O (Fig. 3A). In addition, because injection of the BmFATP dsRNA had no effect on pupal development or on adult emergence, we next injected varying concentrations (1, 5, and 10 μg) of the same dsRNA into 1-day-old female pupae. To assess the effects of BmFATP knockdown on bombykol production, the newly emerged female moths were decapitated, injected with 5 pmol of synthetic PBAN, and the amount of bombykol in the PGs measured (see "Experimental Procedures"). As shown in Fig. 3B, a significant dose-dependent reduction in bombykol production was achieved; injections of 1, 5, and 10 μg dsRNA corresponding to BmFATP elicited 11%, 28%, and 44% reduction, respectively. Because no decrease in bombykol production was observed in the control pupae injected with dsRNA corresponding to enhanced GFP (data not shown), these results indicate that disruption of bombykol production was specific to the dsRNA sequence. RNAi-mediated Knockdown of BmFATP Transcripts in Vivo: Effect on Cytoplasmic LD Dynamics—The most obvious characteristic feature of bombykol-producing cells is the abundance of LDs within the cytoplasm (4Fónagy A. Yokoyama N. Okano K. Tatsuki S. Maeda S. Matsumoto S. J. Insect. Physiol. 2000; 46: 735-744Crossref PubMed Scopus (45) Google Scholar, 13Fónagy A. Yokoyama N. Matsumoto S. Arthropod. Struct. Dev. 2001; 30: 113-123Crossref PubMed Scopus (39) Google Scholar). The role of the LDs in bombykol biosynthesis is to store the bombykol precursor fatty acid in the form of TGs and release it in response to the external signal of PBAN (14Matsumoto S. Fónagy A. Yamamoto M. Wang F. Yokoyama N. Esumi Y. Suzuki Y. Insect. Biochem. Mol. Biol. 2002; 32: 1447-1455Crossref PubMed Scopus (41) Google Scholar). To monitor the LD dynamics during pheromonogenesis, we have previously stained the LDs with the fluorescent lipid marker, Nile Red, and found that they appear 1 to 2 days before eclosion, rapidly accumulate on the day of eclosion, and decrease in size and number after eclosion in response to PBAN (13Fónagy A. Yokoyama N. Matsumoto S. Arthropod. Struct. Dev. 2001; 30: 113-123Crossref PubMed Scopus (39) Google Scholar). Based on these findings, we further examined the effects of BmFATP RNAi on the LD dynamics during pheromonogenesis (Fig. 4). As expected in the non-RNAi-treated control, Nile Red staining revealed that female moths decapitated immediately after eclosion accumulated numerous LDs in the PG, subsequent PBAN injection caused a striking reduction of the size and number in the LDs (Fig. 4, A and B). In contrast, PGs of female moths treated with BmFATP dsRNA accumulated noticeably smaller LDs (Fig. 4C). This result was further confirmed by measuring the total amounts of TGs in the PGs using normal-phase HPLC on a PEGASIL-Silica column (Fig. 5). In the BmFATP knockdown PG, the overall TG content after decapitation was reduced ∼35% compared with the normal PG (Fig. 5, A and C). Taken together, these results indicate that BmFATP plays an essential role in LD accumulatio
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