CYSTEINE RESIDUES OF PHOTORECEPTOR PERIPHERIN RDS - ROLE IN SUBUNIT ASSEMBLY AND AUTOSOMAL-DOMINANT RETINITIS-PIGMENTOSA/

Peripherin/rds is a tetraspanning membrane glycoprotein that is essent ial for the morphogenesis and stabilization of outer segments of verte brate rod and cone photoreceptor cells. Mutations in the ene for perip herin/rds are responsible for retinal degeneration in the rds mouse an d a variety of progressive human retinal degenerative diseases includi ng: autosomal dominant retinitis pigmentosa and macular dystrophy. Per ipherin/rds associates with rom-1. a homologous subunit, to form a het erotetrameric complex. This study examines the importance of cysteine residues fur the structure of peripherin/rds and its assembly with rom -1. Each of the 13 cysteine residues iu bovine peripherin/rds was indi vidually replaced with a serine residue by site-directed mutagenesis: and the resulting mutants were expressed individually or together with rom-1 in COS-I cells. SDS-polyacrylamide gel electrophoresis, immunop recipitation, and velocity sedimentation were carried out to evaluate the ability of these mutants to form disulfide-linked homodimers, asso ciate with rom-1, and assemble into tetramers characteristic of wild-t ype peripherin/rds. Substitution of each of the six nonconserved cyste ines had no apparent effect on dimer formation, folding, or subunit as sembly. In contrast, replacement of any of the seven conserved cystein e residues predicted to lie within a 150 amino acid intradiscal loop s ignificantly altered these properties. Six of these mutants, including a C214S mutant linked to autosomal dominant retinitis pigmentosa: wer e unable: to fold normally, interact with rom-1, or self-assemble into tetramers but instead formed a mixture of large aggregates and a smal ler component, most Likely a dimer. The C150S mutant, on the other han d, was incapable of forming intermolecular disulfide bonds but did ass ociate with rom-1 into a heterotetramer. These results. suggest that ( 1) the conserved C150 residue is required for intermolecular disulfide bonding but not subunit assembly; (2) the six other conserved cystein e residues are crucial for proper folding and subunit assembly, possib ly through formation of intramolecular disulfide bonds; and (3) the mi sfolding and defective subunit assembly of the C214S mutant is respons ible for a form of monogenic autosomal dominant retinitis pigmentosa.