ABSTRACT
Phototransduction in Drosophila is a phosphoinositide-mediated signalling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signalling. cds mutants develop light-induced retinal degeneration. We report here the isolation and characterization of two murine genes encoding this enzyme, Cds1 and Cds2. The genes encode proteins that are 73% identical and 92% similar but exhibit very different expression patterns. Cds1 shows a very restricted expression pattern but is expressed in the inner segments of the photoreceptors whilst Cds2 shows a ubiquitous pattern of expression. Using fluorescent in situ hybridization we have mapped Cds1 and Cds2 to chromosomes 5E3 and 2G1 respectively. These are regions of synteny with the corresponding human gene localization (4q21 and 20p13). Transient transfection experiments with epitope tagged proteins have also demonstrated that both are associated with the endoplasmic reticulum.
Subject(s)
Diacylglycerol Cholinephosphotransferase/genetics , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Chromosome Mapping , Chromosomes, Mammalian/genetics , Cricetinae , Cricetulus , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Diacylglycerol Cholinephosphotransferase/metabolism , Endoplasmic Reticulum/metabolism , Exons , Female , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Genes/genetics , In Situ Hybridization, Fluorescence , Introns , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Molecular Sequence Data , Plasmids/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , TransfectionABSTRACT
The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with lambda(max) (the peak of maximal absorbance) values generally between 400 and 430 nm, and ultraviolet-sensitive (UVS), with a lambda(max)<380 nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86-->Tyr substitution is sufficient by itself to shift the lambda(max) of the goldfish pigment from a wild-type value of 360 nm to around 420 nm, and the reverse substitution of Tyr-86-Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.