Cysteine Residues of Photoreceptor Peripherin/rds: Role in Subunit Assembly and Autosomal Dominant Retinitis Pigmentosa

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• 作者：Andrew F. X. Goldberg ; Christopher J. R. Loewen ; and Robert S. Molday
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：January 13, 1998
• 年：1998
• 卷：37
• 期：2
• 页码：680 - 685
• 全文大小：74K
• 年卷期：v.37,no.2(January 13, 1998)
• ISSN：1520-4995

文摘

Peripherin/rds is a tetraspanning membrane glycoproteinthat is essential for the morphogenesisand stabilization of outer segments of vertebrate rod and conephotoreceptor cells. Mutations in the genefor peripherin/rds are responsible for retinal degeneration in therds mouse and a variety of progressivehuman retinal degenerative diseases including autosomal dominantretinitis pigmentosa and maculardystrophy. Peripherin/rds associates with rom-1, a homologoussubunit, to form a heterotetrameric complex.This study examines the importance of cysteine residues for thestructure of peripherin/rds and its assemblywith rom-1. Each of the 13 cysteine residues in bovineperipherin/rds was individually replaced with aserine residue by site-directed mutagenesis, and the resulting mutantswere expressed individually or togetherwith rom-1 in COS-1 cells. SDS-polyacrylamide gelelectrophoresis, immunoprecipitation, and velocitysedimentation were carried out to evaluate the ability of these mutantsto form disulfide-linked homodimers,associate with rom-1, and assemble into tetramers characteristic ofwild-type peripherin/rds. Substitutionof each of the six nonconserved cysteines had no apparent effect ondimer formation, folding, or subunitassembly. In contrast, replacement of any of the seven conservedcysteine residues predicted to lie withina 150 amino acid intradiscal loop significantly altered theseproperties. Six of these mutants, includinga C214S mutant linked to autosomal dominant retinitis pigmentosa, wereunable to fold normally, interactwith rom-1, or self-assemble into tetramers but instead formed amixture of large aggregates and a smallercomponent, most likely a dimer. The C150S mutant, on the otherhand, was incapable of formingintermolecular disulfide bonds but did associate with rom-1 into aheterotetramer. These results suggestthat (1) the conserved C150 residue is required for intermoleculardisulfide bonding but not subunitassembly; (2) the six other conserved cysteine residues are crucial forproper folding and subunit assembly,possibly through formation of intramolecular disulfide bonds; and (3)the misfolding and defective subunitassembly of the C214S mutant is responsible for a form of monogenicautosomal dominant retinitispigmentosa.