Heterologous expression of bovine rhodopsin in Drosophila photoreceptor cells.
OBJECTIVE
Vertebrate and invertebrate visual pigments are similar in amino acid sequence, structural organization, spectral properties, and mechanism of action, but possess different chromophores and trigger phototransduction through distinct biochemical pathways. The bovine opsin gene (Rho) was expressed in Drosophila, to examine the properties of a vertebrate opsin within invertebrate photoreceptor cells.


METHODS
Transgenic Drosophila expressing the bovine opsin gene (Rho) in photoreceptors were created. Protein expression and cellular location of bovine rhodopsin was assessed by protein blots and immunofluorescence. The glycosylation state was determined by mobility profiles in SDS-PAGE before and after treatment with endoglycosidase. The rhodopsin chromophore was determined by HPLC-mass spectroscopy (MS) and the spectral properties by spectroscopy. The ability of the bovine rhodopsin to couple to Drosophila phototransduction components was assessed by electroretinography and to couple to vertebrate transducin by light-mediated GTPgammaS-binding assays.


RESULTS
Rho showed stable expression even in the absence of endogenous Rh1 opsin and chromophore. It was correctly targeted to the rhabdomeric membranes. Rho remained glycosylated during the maturation process and possessed a distinct glycosylation pattern from that of native Rho. The Drosophila-expressed Rho associated with the 3-hydroxyretinal chromophore but failed to evoke an electroretinogram response from fly photoreceptors. However, the Drosophila-expressed Rho activated transducin in a light-dependent manner.


CONCLUSIONS
Drosophila photoreceptors express a vertebrate rhodopsin as a functional visual pigment, but the expression does not activate the Drosophila phototransduction pathway. The system allows the characterization and comparison of vertebrate and invertebrate visual pigment properties in a common cell type.
Ahmad
Syed Tariq
ST
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556-0369, USA.
Natochin
Michael
M
Barren
Brandy
B
Artemyev
Nikolai O
NO
O'Tousa
Joseph E
JE
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004596:Electroretinography; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D008401:Gas Chromatography-Mass Spectrometry; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors; D006031:Glycosylation; D055537:Light Signal Transduction / Q000502:physiology; D030781:Organisms, Genetically Modified; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics*; D015293:Transducin / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes / Q000502:physiology*
D012243:Rhodopsin; D015293:Transducin