Loewen C J, Moritz O L, Molday R S
Department of Biochemistry and Molecular Biology and Department of Ophthalmology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2001 Jun 22;276(25):22388-96. doi: 10.1074/jbc.M011710200. Epub 2001 Apr 10.
Peripherin-2 and Rom-1 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-1 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-1 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-1 to form tetramers and higher order disulfide-linked oligomers characteristic of the wild-type proteins. The G113E Rom-1 mutant expressed 20-fold lower than wild-type Rom-1, indicating that it behaves mechanistically as a null allele. We conclude that Leu(185) of peripherin-2 (Leu(188) of Rom-1) is critical for tetramer but not dimer formation and that the core tetramer has 2-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.
外周蛋白-2和Rom-1是同源的四次跨膜蛋白，它们组装成非共价四聚体和参与光感受器盘形态发生的高阶二硫键连接的寡聚体。同时遗传L185P外周蛋白-2突变和无效或G113E Rom-1突变的个体患有色素性视网膜炎，而仅遗传一个缺陷基因的个体是正常的。我们通过速度沉降、共免疫沉淀和交联研究了L185P和L185A外周蛋白-2以及L188P Rom-1的表达、亚基组装和二硫键介导的寡聚化。这些突变体在二硫键还原条件下形成非共价二聚体，但未能组装成核心四聚体。在非还原条件下，L185P二聚体形成二硫键连接的四聚体，但不形成高阶寡聚体。L185P与野生型外周蛋白-2和Rom-1共组装形成野生型蛋白特有的四聚体和高阶二硫键连接的寡聚体。G113E Rom-1突变体的表达比野生型Rom-1低20倍，表明其在机制上表现为无效等位基因。我们得出结论，外周蛋白-2的Leu(185)（Rom-1的Leu(188)）对四聚体形成至关重要，但对二聚体形成不重要，并且核心四聚体具有二重对称性。含外周蛋白-2的四聚体是高阶二硫键连接的寡聚体形成所必需的。这些寡聚体的水平对于稳定的光感受器盘形成和双基因色素性视网膜炎疾病表型至关重要。
