The Drosophila ninaG Oxidoreductase Acts in Visual Pigment Chromophore Production

Abstract

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaGP330 mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaGP330. These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal. The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaGP330 mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaGP330. These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal. Vitamin A provides the retinal chromophores that mediate the sensing of light quanta by visual pigments. Vitamin A is a fat-soluble vitamin that cannot be synthesized de novo by most animals. Rather vitamin A is obtained from the diet, either as preformed vitamin A such as retinol from animal sources, or as provitamin A carotenoids from plant sources. These dietary forms must be both transported to the retina and chemically modified to play a key role in visual transduction (1Blomhoff R. Green M.H. Berg T. Norum K.R. Science. 1990; 250: 399-404Crossref PubMed Scopus (522) Google Scholar, 2Yeum K.J. Russell R.M. Annu. Rev. Nutr. 2002; 22: 483-504Crossref PubMed Scopus (441) Google Scholar). Drosophila nina mutations have provided some insights into processing of vitamin A for use in vision. Eight Drosophila nina genes are known (3Pak W.L. Investig. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar). These nina mutants, named for the “neither inactivation nor afterpotential” electroretinogram defect, all possess low rhodopsin levels responsible for this phenotype. Vitamin A-deprived flies also show the same electroretinogram defects and low rhodopsin levels (4Harris W.A. Ready D.F. Lipson E.D. Hudspeth A.J. Stark W.S. Nature. 1977; 266: 648-650Crossref PubMed Scopus (93) Google Scholar, 5Ozaki K. Nagatani H. Ozaki M. Tokunaga F. Neuron. 1993; 10: 1113-1119Abstract Full Text PDF PubMed Scopus (71) Google Scholar), establishing the importance of vitamin A availability in the production of rhodopsin. The ninaB and ninaD mutants are also known to lower rhodopsin levels by disrupting vitamin A availability. The ninaD gene encodes a scavenger receptor (6Kiefer C. Sumser E. Wernet M.F. Von Lintig J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10581-10586Crossref PubMed Scopus (205) Google Scholar) required for efficient use of carotenoids, such as β-carotene, from the diet. The ninaB encodes an oxygenase enzyme (7von Lintig J. Dreher A. Kiefer C. Wernet M.F. Vogt K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1130-1135PubMed Google Scholar) responsible for cleaving β-carotene to generate retinal. Both of these gene products act outside of the retina to carry out their roles in vitamin A metabolism (8Gu G. Yang J. Mitchell K.A. O'Tousa J.E. J. Biol. Chem. 2004; 279: 18608-18613Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Five different retinoids are known to serve as visual pigment chromophores within the animal kingdom. These are retinal, (3,4)-didehydroretinal, (3R)-3-hydroxyretinal, (3S)-3-hydroxyretinal, and (3R)-4-hydroxyretinal. Although the 3R enantiomer of 3-hydroxyretinal is found among many insect orders, the 3S enantiomer is found only among the Cyclorrhapha suborder of Diptera (9Seki T. Vogt T.F. Comp. Biochem. Physiol. B. 1998; 119: 53-64Crossref Scopus (43) Google Scholar) that includes Drosophila (10Seki T. Isono K. Ito M. Katsuta Y. Eur. J. Biochem. 1994; 226: 691-696Crossref PubMed Scopus (19) Google Scholar). The biochemical pathways responsible for production of this retinoid and the other modified chromophores from retinal or other vitamin A precursors have not been characterized. Here we describe the molecular characterization and phenotypic analysis of ninaG. The ninaG gene encodes a member of the glucose-methanol-choline (GMC) 1The abbreviations used are: GMC, glucose-methanol-choline; PDA, prolonged depolarizing afterpotential; HPLC, high performance liquid chromatography; MS, mass spectroscopy; GFP, green fluorescent protein.1The abbreviations used are: GMC, glucose-methanol-choline; PDA, prolonged depolarizing afterpotential; HPLC, high performance liquid chromatography; MS, mass spectroscopy; GFP, green fluorescent protein. family of oxidoreductases (11Lamark T. Kaasen I. Eshoo M.W. Falkenberg P. McDougall J. Strom A.R. Mol. Microbiol. 1991; 5: 1049-1064Crossref PubMed Scopus (210) Google Scholar). The biochemical substrates of most of these enzymes are not known, but some of the enzymes are dehydrogenases and oxidases carrying out redox reactions on a wide variety of organic metabolites. We show that NinaG is required for the biogenesis of the Drosophila rhodopsin chromophore, (3S)-3-hydroxyretinal. This study provides the first molecular description of an enzyme active in modification of retinal to an alternative visual pigment chromophore. ninaG Mutant Analysis—The ethylmethane sulfonate-induced ninaGP330 strain was obtained from W. L. Pak, Purdue University (3Pak W.L. Investig. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar). ninaG1 was identified following an ethylmethane sulfonate screen done in the O'Tousa laboratory and ninaG2 was identified within an existing laboratory stock. Deficiency chromosomes were obtained from the Drosophila Stock Center, Bloomington, Indiana and W. Engels, University of Wisconsin. Drosophila strains were maintained at room temperature and usually reared on standard cornmeal-molasses medium. Pseudopupil and protein blots assessment of rhodopsin content was carried out as described in Hsu et al. (12Hsu C.D. Adams S.M. O'Tousa J.E. Vision Res. 2002; 42: 507-516Crossref PubMed Scopus (7) Google Scholar). Electroretinogram and electron microscopic analyses were performed as described previously (13Washburn T. O'Tousa J.E. Genetics. 1992; 130: 585-595Crossref PubMed Google Scholar), in both cases using 2-day-old flies maintained in 12-h light/12-h dark light cycle. Rhodopsin cellular location was visualized using transgenic flies carrying pRh1:Rh1-GFP, constructed using the Rh1-GFP coding sequence described by Pichaud and Desplan (14Pichaud F. Desplan C. Development. 2001; 128: 815-826PubMed Google Scholar) and the Rh1 promoter region. 2K. Hibbard and J. E. O'Tousa, unpublished data. Ommatidia of newly eclosed Drosophila carrying this element in appropriate genetic backgrounds were prepared by modification of described protocols (15Hardie R.C. Voss D. Pongs O. Laughlin S.B. Neuron. 1991; 6: 477-486Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 16Hardie R.C. J. Neurosci. 1991; 11: 3079-3095Crossref PubMed Google Scholar). The dispersed ommatidia were immersed in a drop of chilled phosphate-buffered saline, pH 7.4, on a glass slide. To-Pro-3 iodide (Molecular Probes) was added to visualize nuclei, anti-fade reagent (Molecular Probes) was added in equal volume, and the GFP fluorescence was then imaged with Bio-Rad MRC 1024 Scanning Confocal microscope. Vitamin A-free medium was prepared as described by Nichols and Pak (17Nichols R. Pak W.L. Drosoph. Inf. Serv. 1985; 61: 195Google Scholar). To generate flies deficient in vitamin A, a life cycle (from egg to adult) was completed on this medium. Replenishment with all-trans-retinal (Sigma) was done by placing adult flies on a supplemented medium for 3 days prepared by adding 10 μl of a 10 mm solution/ml of vitamin A-free medium. For Rh1 RNA analysis, adult flies were flash-frozen in liquid nitrogen and vortexed to separate the heads from the bodies. Total RNA was isolated from different fly strains using the RNeasy mini kit (Qiagen). 15 μg of total RNA was subjected to 1.2% formaldehyde-agarose gel electrophoresis. 18 and 28 S ribosomal RNA standards were visualized on the same gel with ethidium bromide staining to evaluate RNA integrity and as loading controls. The gel was blotted onto Hybond-XL nylon transfer membrane (Amersham Biosciences) using the manufacturer's instructions, probed with Rh1 cDNA, random-labeled with [α-32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences) and autoradiographed to visualize Rh1 RNA levels. FRT/FLP (flippase recombinase/flippase recombinase target) genetic mosaics were constructed and analyzed as described previously (8Gu G. Yang J. Mitchell K.A. O'Tousa J.E. J. Biol. Chem. 2004; 279: 18608-18613Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). ninaG Molecular Analysis—For sequence analysis of the 15 genes in the ninaG region, 1 μl of genomic DNA, prepared using the fly DNA Genetics. 1993; PubMed Google Scholar), was used in with DNA High and to a from the region. The products were into the were using and and on For transgenic of a from of the ninaG promoter region, the open reading and of the region was into the element PubMed Scopus Google Scholar) and into the ninaGP330 mutant using standard Drosophila a Scholar). and for retinal was from the of et al. T. Y. Y. S. Vision Res. PubMed Scopus Google Scholar). Drosophila were in liquid and the heads were from by in μl of and at for of was then the was and at for 10 1 of was and at for The organic was the was with and and the organic were to were in μl of the and then a to A system with a and with a mass was used for liquid were from to and mass were the mass at a of A was onto a and a The of for 3 a to and of at a of 1 The was to into the of the mass and was added to all as a of for The ninaG Mutant of mutants for “neither inactivation nor afterpotential” were identified on the of their electroretinogram O'Tousa J. Pak W.L. Biol. 36: Google Scholar). a a that of the This is to as the prolonged depolarizing The of the in nina mutants, such as for ninaGP330 in is because of low levels of Rh1 rhodopsin within the (3Pak W.L. Investig. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar, J.E. W. J. Pak W.L. 1985; Full Text PDF PubMed Scopus Google Scholar). The ninaG mutant low levels of Rh1 rhodopsin when in protein blots The for ninaG in production of other was by protein blots the Rh4 and the Rh6. show that Rh4 and production not ninaG results were for other ninaG ninaG is only required for efficient biogenesis of a of Drosophila visual pigments. To of other is in ninaG mutants, and protein levels were is a that with rhodopsin in the E. K. K. Full Text Full Text PDF PubMed Scopus Google Scholar). is a membrane protein that is to the M. S. O'Tousa J.E. J. Biol. 1993; PubMed Scopus Google Scholar). nor protein levels are in ninaG mutants lower analysis was used to ninaG was required for of Rh1 levels of of the Rh1 was by analysis in and ninaG2 heads from the the were used as the These results show that ninaG mutants do not affect Rh1 establishing that ninaG acts in or of rhodopsin Rh1 in ninaG the of the ninaG on the of Rh1 rhodopsin in the we flies by the Drosophila Rh1 The location of Rh1-GFP was visualized in of isolated the of the the the Rh1-GFP to the of ninaG mutants, Rh1-GFP to the to the The of Rh1-GFP in ninaG mutants is to that observed in vitamin A-deprived flies not the ninaGP330 a in the of the and is when with We also observed of the endoplasmic reticulum in the and results were observed in ninaG2 not The ninaGP330 mutant these defects at all with other retinal observed to days of not The the of ninaG NinaG is required within we genetic mosaics in only the was mutant for ninaG. 3 the rhodopsin content in ninaG mosaics and an appropriate of the ninaG the retinal is mutant for but the of the animal is These flies show low levels of rhodopsin, that NinaG is required within or other of the retina for rhodopsin of the ninaG the of ninaG as a first and the ninaG gene. for ninaG and of a of of the of the were for rhodopsin content using the of the dark R. Google Scholar). The of this in flies is of high rhodopsin levels. and and flies dark of low rhodopsin levels. rhodopsin levels in these were by analysis not This analysis placed the location of ninaG within region on the of the To this within the region K. Science. 2001; PubMed Scopus Google Scholar) were for with ninaG and but not to the low rhodopsin of ninaG These results placed ninaG into a small region by the gene and by the gene. of and ninaG. ninaG is to in the region and This is a genomic region of 15 To the gene responsible for the ninaG we the coding of the 15 identified genes within the region from ninaGP330. different products of to all The coding within these products were with the Drosophila sequence S. M. R.C. M. C. G. A. C. D. R.M. A. J. L. D. S. D. J. P. P. H. E. A. I. S. C. P. de B. A. I. S.M. K. M. S. P. K.J. C. S. W. C. K. A. F. P. M. D. J. D. K.A. M.H. C. M. F. K.A. C.D. C. S. D. P. Y. J. Y. B. D. G. C. J. A. S.M. M. B. L. K.A. K. M. S. J. K. K. F. H. I. M. T. E. M. R. E. R. C. R. E. J. S.M. T. D. Yang S. J. M. G. L. W. S. Science. PubMed Scopus Google Scholar, B. A. S. Adams M. M. E. A. T. S. S. R. K. M. C. G. 2002; Scholar). The gene CG6728 a in the ninaGP330 mutant a to at This in a a codon to a other mutations in mutations were other genes in a of the sequence analysis, CG6728 as the for the ninaG gene. a of the gene the of the found in ninaGP330. We used a genomic that this open reading and of to transgenic that this CG6728 genomic the low rhodopsin of ninaGP330 Further, electroretinogram analysis that the CG6728 the in ninaG mutants These results that CG6728 is the ninaG gene. NinaG a of of the NinaG protein sequence with protein that NinaG is a member of the oxidoreductase This is a family of flavin adenine are 15 identified Drosophila genes in this Y. Mol. Biol. 1992; Google Scholar), for a characterized the gene M. Mol. Biol. Google Scholar) of the Drosophila oxidoreductase family and characterized oxidoreductase enzymes from other The analysis high within this family in and the NinaG protein in results of or from this of analysis that the region of NinaG a sequence This is to a in NinaG protein with and G. Res. PubMed Scopus Google Scholar). The other of the family of the NinaG protein are as for the oxidoreductase the described as the J. J. Mol. Biol. PubMed Scopus Google Scholar) within the region. NinaG in of NinaG as a member of the oxidoreductase family that the NinaG is an enzyme responsible for a redox on small organic molecules. that the Rh1 in the endoplasmic reticulum observed in ninaG mutants is to other and vitamin A-free deficient in rhodopsin we that the NinaG enzyme acts in the production of the Rh1 chromophore. To this was used to profile the retinal from and ninaG mutant A of heads were used for of these and the results were in prepared of these at and These have been to the same to of the were observed among at and the of all were To that all observed are because of retinoid flies were reared on a vitamin A-free diet. from such flies in the region of at the retinal to these adult flies the profile observed for flies reared on medium not that flies possess a major retinoid with a of with a by the at The ninaG flies at and that possess lower levels of both of these The of both the and was and the mass not of an at for of 3-hydroxyretinal. These were as and in with these are the retinoids from Drosophila heads (10Seki T. Isono K. Ito M. Katsuta Y. Eur. J. Biochem. 1994; 226: 691-696Crossref PubMed Scopus (19) Google Scholar). from flies in the dark and provided with all-trans-retinal as the only retinoid a in the at the of the not This is in with the of the because flies light to 3-hydroxyretinal from the to the K. T. Y. Y. J. Physiol. PubMed Scopus Google Scholar). the at for and ninaG The retinoid at and a of the mass of the an at not is the for of retinal. The results of the all-trans-retinal standard a at with a of and not Although the of an at that the is an of retinal, the major in and to the all-trans-retinal standard not this the of the 3-hydroxyretinal from is to Rh1 rhodopsin, the of 3-hydroxyretinal in the ninaG mutant be a of ninaG Rh1 rhodopsin protein To this the profile of retinoids in flies expressing Rh4 in of Rh1 to as R. R. K. D. M. J. Neurosci. 1992; PubMed Google Scholar) was we that Rh4 protein is expressed at levels in both and ninaG. High levels of Rh4 were also observed in the flies in both and ninaG mutant Further, a is required for Rh4 as flies on vitamin A-free medium Rh4 not we the retinal of to the of the Rh4 in and the ninaG and show the of the retinoid in the of flies at and from the most a of and a of to in the from the ninaG the is and the is the retinoid at with a of the Rh4 visual pigment use of a different in the ninaG mutant These results are only with a role for ninaG in We have carried out a molecular and analysis of the Drosophila ninaG gene. ninaG mutants have a in the Rh1 rhodopsin because of a to transport newly synthesized rhodopsin from the endoplasmic reticulum to the rhabdomere. The is with the to act as a for Rh1 the system 1991; Full Text PDF PubMed Scopus Google Scholar). to the this is by ninaD mutants, and vitamin A-deprived flies the retinoid chromophore. The newly synthesized protein must with retinoid and use light to the to the for rhodopsin to from the endoplasmic reticulum and the pathway K. Nagatani H. Ozaki M. Tokunaga F. Neuron. 1993; 10: 1113-1119Abstract Full Text PDF PubMed Scopus (71) Google Scholar). We show that a genomic the CG6728 coding sequence by the Drosophila the mutant rhodopsin of that CG6728 is the ninaG gene. The NinaG protein is a member of the oxidoreductase family J. Mol. Biol. 1992; PubMed Scopus Google Scholar), a of that flavin adenine as a This family from from K.R. J. A. S. S. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), from P. C. K. J. G. 5: PubMed Scopus Google Scholar), and from (11Lamark T. Kaasen I. Eshoo M.W. Falkenberg P. McDougall J. Strom A.R. Mol. Microbiol. 1991; 5: 1049-1064Crossref PubMed Scopus (210) Google Scholar). A with the oxidoreductase family provided on the in vivo role of Although all characterized oxidoreductases act on small organic act on a wide of substrates such as and family have been identified by but their is not Drosophila as the rhodopsin (10Seki T. Isono K. Ito M. Katsuta Y. Eur. J. Biochem. 1994; 226: 691-696Crossref PubMed Scopus (19) Google Scholar). of from retinal or retinoid both hydroxylation and either of be by the NinaG oxidoreductase. The the 3R and 3S forms of 3-hydroxyretinal is because of a at the and Vogt (9Seki T. Vogt T.F. Comp. Biochem. Physiol. B. 1998; 119: 53-64Crossref Scopus (43) Google Scholar) that be from of such as by and is that be in this of only in the not the use of retinal in visual (9Seki T. Vogt T.F. Comp. Biochem. Physiol. B. 1998; 119: 53-64Crossref Scopus (43) Google Scholar). These the high of the enzyme retinal from β-carotene is responsible for retinal the chromophore. This that the enzymes in that use 3-hydroxyretinal act on but the of Drosophila for production also to act only on β-carotene (7von Lintig J. Dreher A. Kiefer C. Wernet M.F. Vogt K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1130-1135PubMed Google Scholar, Lintig J. Vogt K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These are with the that the following are required for use of and in the are to β-carotene, β-carotene is then to generate retinal, the retinal is to the and retinoid used in some animals. To the NinaG oxidoreductase acts in we characterized the retinoid content in the ninaG mutant using high performance liquid is a in 3-hydroxyretinal in ninaG mutants, that ninaG mutants are in is the that the Rh4 rhodopsin a different in the and ninaG Rh4 rhodopsin as 3-hydroxyretinal. the ninaG Rh4 a chromophore, from 3-hydroxyretinal by and mass These results are by a in the production of 3-hydroxyretinal in the ninaG The results that Rh1 is in the use of 3-hydroxyretinal as a chromophore, Rh4 this alternative chromophore. The of this alternative is but an or of alternative in the of NinaG the Rh1 visual pigment is characterized by a of a light in to the for the Rh1 protein. This light is because of the of a retinoid pigment that is from the visual pigment chromophore. This pigment is to in the light and transfer this to the Rh1 K. Nature. 1977; PubMed Scopus Google Scholar). The in the suborder Cyclorrhapha are the same of the (9Seki T. Vogt T.F. Comp. Biochem. Physiol. B. 1998; 119: 53-64Crossref Scopus (43) Google Scholar). the use of be to biogenesis of Rh1 but not to the biogenesis of other visual pigments. The results show that the NinaG protein acts within the biochemical pathways responsible for the of (3S)-3-hydroxyretinal. The biochemical in this are not The of a in of was previously in Drosophila T. Isono K. Ozaki K. Y. Y. Ito M. T. M. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar), but an to not been The NinaG protein act in this within a pathway responsible for that the 3-hydroxyretinal in the ninaG mutant is the 3R an for the inability of this 3-hydroxyretinal to serve as the Rh1 chromophore. of β-carotene to retinal outside the retina (8Gu G. Yang J. Mitchell K.A. O'Tousa J.E. J. Biol. Chem. 2004; 279: 18608-18613Abstract Full Text Full Text PDF PubMed Scopus (35) Google retinal or other must be transported to the retina for use as the visual The of ninaG the of 3-hydroxyretinal biogenesis within the The of a sequence in the NinaG protein that the protein is to the endoplasmic the of the endoplasmic reticulum be to the NinaG oxidoreductase for endoplasmic reticulum of NinaG is that newly synthesized Rh1 protein to be a to the chromophore. This to within the for of the visual as the Rh1 rhodopsin also with a chromophore, the is that efficient biogenesis of visual of the and We the following for and strains used in this Pak, the ninaGP330 Engels, in the ninaG the the and and and We for with the DNA sequence Adams and Mitchell for with genetic and and and Yang for of this we are to the for and at the University of for to the and mass