Molecular characterization of peripherin-2 and Rom-1 mutants responsible for digenic retinitis pigmentosa

Peripherin-2 and Rom-l are homologous tetraspanning membrane proteins that assemble into noncovalent tetramers and higher order disulfide-linked oligomers implicated in photoreceptor disc morphogenesis. Individuals who coinherit a L185P peripherin-2 mutation and a null or G113E rom I mutation are afflicted with retinitis pigmentosa, whereas individuals who inherit only one defective gene are normal. We examined the expression, subunit assembly, and disulfide-mediated oligomerization of L185P and L185A peripherin-2 and L188P Rom-l by velocity sedimentation, co-immunoprecipitation, and cross-linking. These mutants formed noncovalent dimers under disulfide-reducing conditions but failed to assemble into core tetramers. Under nonreducing conditions, L185P dimers formed disulfide linked tetramers but not higher order oligomers. L185P coassembled with wild-type peripherin-2 and Rom-l to form tetramers and higher order disulfide-linked oligomers characteristic of the wild-type proteins. The G113E Rom-l mutant expressed 20-fold lower than wildtype Rom-l, indicating that it behaves mechanistically as a null allele. We conclude that Leu(185) of peripherin-2 (Leu(188) of Rom-l) is critical for tetramer but not dimer formation and that the core tetramer has a-fold symmetry. Peripherin-2-containing tetramers are required for higher order disulfide-linked oligomer formation, The level of these oligomers is critical for stable photoreceptor disc formation and the digenic retinitis pigmentosa disease phenotype.