Molecular characterization of the skate peripherin/rds gene: relationship to its orthologues and paralogues.

The outer segments of vertebrate rod and cone photoreceptors are composed of an ordered array of membranous discs that serve as the site for phototransduction. In rods, with the exception of a few nascent discs located at the base of the outer segment, the discs are surrounded by a separate plasma membrane.1 In cones, the discs appear as a folded system of membranes that are continuous, both with each other and with the plasma membrane. Peripherin/rds, a transmembrane glycoprotein, has been localized to the rim region of mature discs and at the basal region adjacent to the cilia of rod and cone outer segments where disc morphogenesis occurs.2 It is proposed to be involved in maintenance of the flattened disc morphology and fusion of membranes, to form isolated discs.3 This is suggested by its location on the disc membrane as well as by the phenotype of retinal degeneration slow (rds) mutants. In rds/+ mice, the phenotype is a semidominant trait characterized by haploinsufficiency of peripherin/rds arising from insertion of a 9.2-kb repetitive genomic element within exon 2 of the gene.4 It has been shown that the rds defect in rds/+ retina is more deleterious to rods than cones.5 The outer segment membrane biogenesis occurs in the rds/rds but the membrane is unable to fold into the proper disc structure. Peripherin/rds cDNA has been isolated and sequenced from human,6 cow,7 dog,8 cat,9 rat,10 mouse,11 chicken (CRDS1 and CRDS2),12 and frog (XRDS35, XRDS36, and XRDS38)13 and found to code for proteins ranging in length from 346 to 364 residues, depending on the species. The predicted polypeptide is composed of four putative transmembrane segments, relatively small (21 residues) and large (142 residues) intradiscal loops, and a long C-terminal segment exposed to the cytoplasmic side of the disc membranes (for review, see Ref. 14). In vitro biochemical studies suggested a noncovalent association between peripherin/rds and ROM-1,14–18 a nonglycosylated transmembrane protein that shares several characteristics with peripherin/rds. These characteristics include similar hydropathy profiles, gene organization, highly conserved residues, and localization to the rim region of rod and cone outer segments.14, 15, 19 Both in vivo and in vitro studies have shown that the noncovalent interactions between peripherin/rds and ROM-1 act to form homomeric and heteromeric functional core complexes. Although proper assembly between peripherin/rds and ROM-1 is believed to play a crucial role in normal outer segment structure, the functional activities and the site of interactions between the two proteins at the molecular level are not completely understood. Goldberg et al.17 used site-directed mutagenesis to determine the role of cysteine residues of peripherin/rds in the functional core complex formation. The mouse peripherin/rds has 13 cysteine residues and 11 of them are conserved in all known peripherin/rds. Sequence comparison with ROM-1, however, shows that only seven of these cysteines located in the large intradiscal loop are conserved. Some or all these cysteine residues may form intra- or intermolecular disulfide bonds. Replacement of the noncon-served cysteines showed no apparent effect on dimer formation, folding, or subunit assembly. In contrast, replacement of any of the seven conserved cysteine residues within the large intradiscal loop significantly alters these properties,17 suggesting that these residues are crucial for proper folding and subunit assembly. Furthermore, the carboxyl terminus of pe-ripherin/rds has been shown to promote membrane fusion in vitro, signifying a possible role for this protein in outer segment renewal.3,20,21 Recently, peripherin/rds has been shown to associate with the photoreceptor cGMP-gated channel Na/Ca-K exchanger.22 It has been suggested that the glutamic acid–rich protein of the channel may act as a bridge to connect the channel-exchanger complex with peripherin/rds.22 This association may play a role in connecting the rim region of the disc to the plasma membrane and/or anchoring the cGMP-gated channel in the plasma membrane. Interest in peripherin/rds has increased since the discovery of its association with different forms of human retinal diseases. More than 80 different pathogenic mutations have been identified that are associated with retinitis pigmentosa (RP) and several forms of macular dystrophy (MD; for review, see Refs. 23–25). These mutations include base substitutions that cause missense mutations or premature termination and in-frame insertion/deletion mutations that change the reading frame. The majority of these mutations are located in the large intradiscal loop, emphasizing the important role played by this region in the function of peripherin/rds. Most of our knowledge of the structure and function of peripherin/rds has come from studies of mammalian genes. In the present study, we determined the structure of the skate peripherin/rds (srds) gene, the sequence of the intron/exon boundaries, and the sequence analysis of the cDNA clones. We studied the evolution and structural properties of srds by comparing the skate gene with homologues from human, cow, dog, cat, mouse, rat, chicken, and the African clawed frog. We provide evidence that there are highly conserved regions in all peripherin/rds proteins, suggesting important functional roles for these domains.