Glycosylation and palmitoylation are not required for the formation of the X-linked cone opsin visual pigments.

The visual pigments are a family of photoreceptor pro-teins that absorb light and mediate vision [1-7]. Humans havetwo groups of visual pigments, rhodopsin, which is expressedin rod cells and provides monochromatic vision under lowintensity light, and the cone opsins, which provide color vi-sion under higher intensity light. There are three cone opsinpigments with short (S cone or blue), medium (M cone orgreen), or long (L cone or red) wavelength absorption spectra,each encoded by a separate gene [8]. The genes for red andgreen cone opsins are found in a tandem array on the distallong arm of the X chromosome, which may contain varyingcopy numbers of red, green and red/green hybrid genes; theblue opsin gene is a single-copy locus located at 7q22-qter [8-10]. All of the visual pigments are composed of an apoproteinmolecule (or opsin) that is conjugated to the chromophore,11-cis-retinal. In response to the absorption of a photon oflight, the chromophore is isomerized to all- trans -retinal. Thisconformational change in the visual pigment molecule causesactivation of the G-binding protein, transducin.The structure and function of the rhodopsin molecule havebeen extensively studied by peptide mapping and site-directedmutagenesis and is predicted to be a protein with seven trans-membrane segments. The cone opsins share 40-44% homol-ogy with rhodopsin at the amino acid level, suggesting simi-lar structures (The structure for the green opsin gene is shownin Figure 1) [8]. Regions of structural similarity between theX-linked cone opsins and rhodopsin include a lysine at posi-tion 312, which is predicted to form a Schiff base with retinal,and cysteines at residues 126 and 203 that function as sites ofdisulfide cross-linking [11-15]. The comparable sites inrhodopsin are a lysine at position 296 and cysteines at resi-dues 110 and 187 [12].Previously it has been shown that both rhodopsin and theX-linked cone opsins are N-linked glycosylated and thatrhodopsin is palmitoylated [16-20]. Rhodopsin is glycosylatedat residues N2 and N15, although glycosylation only at aspar-agine-15 is required to form a visual pigment. Here, we showthat elimination of the N-linked glycosylation does not impairthe ability of green opsin to form a visual pigment and that thewild-type green opsin is not palmitoylated.METHODSPlasmids: Recombinant plasmids included a full-length, syn-thetic bovine rhodopsin cDNA (pRho), a mutant bovinerhodopsin cDNA (pRhoC187R), and a full-length green cDNA(pGrn) that included the epitope for the C-terminus of rhodop-sin [15,16,21]. Alteration of the N-linked glycosylation sitefrom asparagine to serine at residue 32 (N32S, see Figure 1)was performed by site-directed mutagenesis (Transformer Site-Directed Mutagenesis Kit, Clontech Laboratories, Palo Alto,CA). The selection primer for transforming a MluI site to aHindIII site was 5'-CGACGGTATCGATAAGCTTGATATCGAATTCC-3'. The primer for mutagenesis (N32S) was 5'-CTCTGGTGGACTGGCTGTTGGTG-3'. All mutants wereverified by sequencing. Recombinant cDNAs were subclonedinto the vector, pMEP4 β (provided by Dr. Mark Tyckocinski,Case Western Reserve University, Cleveland, OH), for cre-